JP6866345B2 - Compounds targeting IL-23A and B cell activating factor (BAFF) and their use - Google Patents

Description

Mutual reference to related applications This application is a US provisional application No. 62 / 196,170 filed July 23, 2015; the same No. 62 / 201,067 filed August 4, 2015; and June 2016. No. 62 / 355,302 of the application filed on the 27th, both claiming the benefit of the filing date under Article 119 of the US Patent Law for the title "COMPOUND TARGETING IL-23A AND B-CELL ACTIVATING FACTOR (BAFF) AND USES THEREOF". And all of them are incorporated herein by reference in their entirety.

Inflammation is associated with the innate and adaptive immune responses required to combat infection. However, if inflammation is not suppressed, autoimmune or autoimmune diseases, neurodegenerative diseases, and even cancer can develop. Inflammatory cytokines such as IL1, BAFF, TNF-α, IL6, IL12, IL17, IL18, and IL23 are known to be involved in inflammation and specific pathways that activate immune cells. It is unclear whether or how inhibition of one or more of the cytokines in the family can treat autoimmune or auto-inflammatory diseases.

Interleukin 23 (IL23) is a heterodimeric cytokine consisting of two subunits, p40 and p19. The p19 subunit is also called IL-23A. Although the p19 subunit is unique to IL23, the p40 subunit is common to the cytokine IL12. IL23 has attracted attention as an important regulator of pathogenic Th17, γδT, and innate lymphoid cells (ILC), which promote the production of IL17, IL22, and other cytokines that cause inflammation and damage to local tissues. IL23 promotes upregulation of the matrix metalloproteinase MMP9, increases angiogenesis, and reduces CD8 + T cell infiltration, and is therefore implicated in the development of cancerous tumors. Furthermore, IL23 cooperates with IL6 and TGFβ1 to stimulate naive CD4 + T cells and differentiate them into Th17 cells. Upon differentiation, Th17 cells produce IL17, which enhances T cell priming and stimulates the production of pro-inflammatory cytokines such as IL1, IL6, TNF-α, NOS-2, as well as inflammation and disease. It is an pro-inflammatory cytokine that also induces the expression of chemokines that cause the etiology of. IL23 exerts its effect via a cell surface receptor composed of the subunit IL12β1 of the IL12 receptor paired with the unique subunit IL23R. Expression of IL23R is confined to a specific population of immune cells and is found primarily in subsets of T cells (αβ and γδTCR +) and NK cells.

In mice, gene disruption of the IL23p19 gene results in a selective loss of IL23 function with severe deficiency of the T-dependent immune response, including decreased production of antigen-specific immunoglobulins and impaired delayed hypersensitivity response (Ghillardi N, et al. (2004) J. Immunol. 172 (5): 2827-33). Knockout mice deficient in either IL23p40 or IL23p19, or any subunit of the IL23 receptor (IL23R and IL12β1), have severe symptoms in animal models of multiple sclerosis, arthritis, and inflammatory bowel disease. Shows reduced severity. Similar results have been confirmed in EAE using antibodies specific for IL23p19, and a T cell-mediated colitis model has also demonstrated the role of IL23 in these disease environments (Chen Y. et al. (2006) J. Clin. Invet. 116 (5): 1317-26; Elson CO. Et al. (2007) Gastroenterology 132 (7): 2359-70). This highlights the importance of IL23 in chronic inflammation (Langowski et al. (2006) Nature 442 (7101): 461-5; Kikly K, et al. (2006) Curr. Opin. Immunol. 18). (6): 670-5). In addition, elevated IL23 production means that it is a major factor in inflammatory arthritis and inflammatory autoimmune diseases (Adamopoulos et al. (2011) J. Immunol. 187: 593-594; and Langris et al. (2005) J. Exp. Med. 201: 233-240). An association with IL23, its downstream cytokine IL22, and bone formation has been reported in a mouse model system overexpressing IL23 (Sherlock et al. (2012) Nat. Med. 18: 1069-76).

B-cell activating factor (BAFF) is a cytokine belonging to the tumor necrosis factor (TNF) ligand superfamily, and is a receptor BAFF-R (BR3), TACI (transmembrane activator and calcium modulator and cyclophilin ligand interlace). Factors and calcium regulators and cyclophilin ligand interacting factors), and act as ligands for BCMA (B cell maturation antigen). The interaction between BAFF and its receptors elicits signals essential for the formation and maintenance of B cells, which allow B cells to synthesize immunoglobulins in response to invasion by foreign substances. Appropriate BAFF levels in patients help maintain normal immune levels, but inadequate levels can lead to immunodeficiency, and excessive levels can lead to abnormally high antibody production. When a patient exhibits autoimmunity, it produces antibodies against tissues or organs in its own body. Autoimmune diseases, including lupus erythematosus and rheumatoid arthritis, result from excessive BAFF levels in the body. Therefore, it is important to regulate BAFF production to treat patients with these diseases.

BAFF can exist in three forms: membrane binding (mbBAFF), soluble trimer BAFF (sBAFF), and multimer form consisting of 60 BAFF monomers (BAFF60mer). The relative importance of various forms of BAFF on normal and disease physiology has not been fully elucidated. As mentioned above, BAFF binds to three receptors, BAFFR (BR3), TACI, and BCMA. Proliferation-inducing ligands (APRIL), a related member of the TNF receptor ligand family, have been shown to bind to TACI and BCMA with high affinity. In contrast to the high affinity APRIL: BCMA interaction, the BAFF: BCMA interaction is low affinity (1-2 μM) and is not considered to play an important role in vivo (Bossen and Schneider). , 2006).

Soluble BAFF is expressed at high levels in individuals with systemic lupus erythematosus (SLE) and in target organs such as the inflamed kidney. Soluble BAFF acts as an important factor for B cell homeostasis and survival (Kalled et al., 2005; Mackay et al., 2003; Smith and Cancro, 2003; Patche et al., 2004). Autoantibody formation by BAFF-dependent B cells begins at the glomerular basement membrane (GBM) and results in glomerular deposition of ICs on mesangium and stromal tissue within the proximal tubule epithelial cells (PTEC). Such IC deposition causes complement fixation and neutrophil activation, resulting in local renal damage. Inflammatory mediators (eg, IL6, IL8, MCP-1) produced by damaged kidney cells (MC, PTEC, renal fibroblasts, endothelial cells) are immune cells (eg, B cells, T cells, etc.). It promotes the inflammatory cycle by increasing the infiltration of dendritic cells, neutrophils, and macrophages).

The anti-BAFF monoclonal antibody belimumab (Benlysta®) has been demonstrated to be capable of reducing autoantibody formation, providing significant benefits to patients with systemic lupus erythematosus (SLE). However, the efficacy of belimumab in SLE patients is at best moderate and there is ample room for improvement (Furie et al, 2011). Therefore, there is still a need for novel compositions with enhanced efficacy for treating and preventing autoimmune or inflammatory diseases. In addition, identifying more effective treatments should allow dose reductions and, in addition, reduce treatment-related costs.

The present specification provides compounds specific to BAFF and IL23A, compositions containing such compounds, and methods of use and production thereof.

Aspects of the present disclosure relate to compounds comprising a first polypeptide and a second polypeptide.
(A) The first polypeptide is
(I) Light chain variable domain (VL1) of the first immunoglobulin specific for the first target protein;
(Ii) Heavy chain variable domain (VH2) of the second immunoglobulin specific for the second target protein; and (iii) Hinge region, heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3). ) Including
(B) The second polypeptide is
(I) Light chain variable domain (VL2) of the second immunoglobulin specific for the second target protein;
(Ii) Containing a heavy chain variable domain (VH1) of a first immunoglobulin specific for the first target protein.
In this compound
a) The VL1 and VH1 associate to form a binding site that binds to the first target protein.
b) The VL2 and VH2 associate to form a binding site that binds to the second target protein.
c) The heavy chain constant region 2 (CH2) contains tyrosine at position 252 and threonine at position 254 and glutamic acid at position 256 according to the EU index similar to Kabat.
d) The first target protein is BAFF and the second target protein is IL-23A, or the first target protein is IL-23A and the second target protein is BAFF. Yes,
Furthermore, in this compound,
i) The VL1 comprises SEQ ID NO: 2, the VH1 comprises SEQ ID NO: 1, the VL2 comprises SEQ ID NO: 4, the VH2 comprises SEQ ID NO: 3, or ii) the VL1 comprises SEQ ID NO: 4. , The VH1 comprises SEQ ID NO: 3, the VL2 comprises SEQ ID NO: 2, the VH2 comprises SEQ ID NO: 1, or iii) said VL1 comprises SEQ ID NO: 85, said VH1 comprises SEQ ID NO: 84. The VL2 comprises SEQ ID NO: 4, the VH2 comprises SEQ ID NO: 4, or iv) the VL1 comprises SEQ ID NO: 4, the VH1 comprises SEQ ID NO: 3, the VL2 comprises SEQ ID NO: 85, and the said. VH2 comprises SEQ ID NO: 84, or v) said VL1 comprises SEQ ID NO: 87, said VH1 comprises SEQ ID NO: 86, said VL2 comprises SEQ ID NO: 4, said VH2 comprises SEQ ID NO: 3, or vi) The VL1 comprises SEQ ID NO: 4, the VH1 comprises SEQ ID NO: 3, the VL2 comprises SEQ ID NO: 87, the VH2 comprises SEQ ID NO: 86, or vii) the VL1 comprises SEQ ID NO: 89. , The VH1 comprises SEQ ID NO: 88, the VL2 comprises SEQ ID NO: 4, the VH2 comprises SEQ ID NO: 3, or viii) The VL1 comprises SEQ ID NO: 4, the VH1 comprises SEQ ID NO: 3. The VL2 comprises SEQ ID NO: 89, the VH2 comprises SEQ ID NO: 88, or ix) the VL1 comprises SEQ ID NO: 91, said VH1 comprises SEQ ID NO: 90, said VL2 comprises SEQ ID NO: 4, said. VH2 comprises SEQ ID NO: 3 or x) said VL1 comprises SEQ ID NO: 4, said VH1 comprises SEQ ID NO: 3, said VL2 comprises SEQ ID NO: 91, said VH2 comprises SEQ ID NO: 90.

In some embodiments, the first polypeptide further comprises a first linker between the VL1 and the VH2, and the second polypeptide further comprises between the VL2 and the VH1. Includes a second linker. In some embodiments, the first linker or the second linker comprises the amino acid sequence GGGSGGGG (SEQ ID NO: 69). In some embodiments, the first linker and the second linker comprise the amino acid sequence GGGSGGGG (SEQ ID NO: 69). In some embodiments, the first polypeptide further comprises a third linker between the VH2 or VL1 and the hinge region, and the second polypeptide further comprises the VH1 (its C-terminus). A fourth linker is included after the VL2 (terminal) or said VL2 (its C-terminal). In some embodiments, the third linker of the first polypeptide comprises the amino acid sequence GGCGGGGEVAACEKEVAALEKEVAALEKEVAALEK (SEQ ID NO: 82), and the fourth linker of the second polypeptide is the amino acid sequence GGCGGGKVAACKEKVALKEKALEKVA SEQ ID NO: 83) is included. In another embodiment, the third linker of the first polypeptide comprises the amino acid sequence GGCGGGKVAACKEKVAALKEKVAALKEKVAALKE (SEQ ID NO: 83), and the fourth linker of the second polypeptide is the amino acid sequence GGCGGGGEVAACEKEVAALEKEVAALEKEVA. Includes number 82). In some embodiments, the third linker of the first polypeptide comprises the amino acid sequence GGCGGGGEVAACEKEVAALEKEVAALEKEVAALEK (SEQ ID NO: 82) or the amino acid sequence GGCGGGKVAACKEKVAALKEKVAALKEKALKE (SEQ ID NO: 83). In another embodiment, the fourth linker of the second polypeptide comprises the amino acid sequence GGCGGGGEVAACEKEVAALEKEVAALEKEVAALEK (SEQ ID NO: 82) or the amino acid sequence GGCGGGKVAACKEKVAALKEKVAALKEKALKE (SEQ ID NO: 83). In some embodiments, the third linker comprises the amino acid sequence VEPKSC (SEQ ID NO: 72) or the amino acid sequence FNRGEC (SEQ ID NO: 71). In some embodiments, the fourth linker comprises the amino acid sequence FNRGEC (SEQ ID NO: 71) or the amino acid sequence VEPKSC (SEQ ID NO: 72). In some embodiments, the third linker comprises the amino acid sequence VEPKSC (SEQ ID NO: 72) and the fourth linker comprises the amino acid sequence FNRGEC (SEQ ID NO: 71).

In some embodiments, the first polypeptide further comprises (depending on the configuration) a heavy chain constant region 1 domain (CH1) between the VH2 or VL1 and the hinge region, said the second. The polypeptide further comprises (depending on the configuration) a light chain constant region domain (CL) at the C-terminus of the VH1 or VL2, where the CL and CH1 are co-associated by disulfide bonds to form the C1 domain. To do. In some embodiments, the first linker (between the VL1 and the VH2) or the second linker (between the VL2 and the VH1) comprises the amino acid sequence GGGSGGGG (SEQ ID NO: 69). .. In some embodiments, the first linker and the second linker comprise the amino acid sequence GGGSGGGG (SEQ ID NO: 69). In some embodiments, the first polypeptide further comprises (depending on the configuration) a third linker between the VH2 or VL1 and the CH1, and the second polypeptide further contains (depending on the configuration). A fourth linker is included between the VH1 or VL2 and the CL (depending on the configuration). In some embodiments, the third or fourth linker comprises the amino acid sequence LGGGSG (SEQ ID NO: 70). In some embodiments, the third linker and the fourth linker include the amino acid sequence LGGGSG (SEQ ID NO: 70). In some embodiments, the third linker and / or the fourth linker comprises any cysteine residue. In some embodiments, the third linker and / or the fourth linker comprises the amino acid sequence GGCGGGG (SEQ ID NO: 135) or LGGCGGGS (SEQ ID NO: 136).

In some embodiments, the heavy chain constant region 2 (CH2) comprises alanine at position 234 and alanine at position 235 according to EU index numbers similar to Kabat for conventional antibodies.

In some embodiments, the amino acid sequence of said hinge region, said heavy chain constant region 2 (CH2), or said heavy chain constant region 3 (CH3) is derived from IgG1 or IgG4. In some embodiments, the hinge region comprises the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 76). The hinge region of SEQ ID NO: 76 is present, for example, in the polypeptide of SEQ ID NO: 5. In another embodiment, the hinge region comprises the amino acid sequence LEPKSSDKTHTCPPCP (SEQ ID NO: 130). The hinge region of SEQ ID NO: 130 is present, for example, in the polypeptide of SEQ ID NO: 9. In yet another embodiment, the hinge region comprises the amino acid sequence ESKYGPPPCPPCP (SEQ ID NO: 134). The hinge region of SEQ ID NO: 134 is present, for example, in the polypeptide of SEQ ID NO: 13.

In some embodiments, the compound comprises two said first polypeptide and two said said second polypeptide, the two first polypeptides being associated together by at least one disulfide bond. In some embodiments, the compound comprises two said first polypeptide and two said said second polypeptide, the two first polypeptides being associated together by at least one disulfide bond. And each of the first polypeptides associates with one of the second polypeptides by at least one disulfide bond.

In some embodiments,
(I) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 5, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 6, or (ii) the first polypeptide comprises SEQ ID NO: 7. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 8 or (iii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 9 and the second polypeptide comprises. Either the amino acid sequence of SEQ ID NO: 10 is contained, or (iv) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 11 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 12 or (v). ) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 13, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 14, or (vi) the first polypeptide comprises the amino acid of SEQ ID NO: 15. The second polypeptide comprises a sequence and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 16 or (vii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 17 and the second polypeptide has the SEQ ID NO: Eighteen amino acid sequences are included, or (viii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 19, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 20 or (ix) said. The first polypeptide comprises the amino acid sequence of SEQ ID NO: 21, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 22, or (x) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 23. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 24, or (xi) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 25 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 26. The first polypeptide comprises the amino acid sequence, or (xii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 27, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 28, or (xiii) said first. The polypeptide comprises the amino acid sequence of SEQ ID NO: 29 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 30 or (xiv) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 31. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 32, or (xv) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 33 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 34. Or (xvi) said first The polypeptide comprises the amino acid sequence of SEQ ID NO: 35 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 36, or (xvii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 37 and said. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 38, or (xviii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 39 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 40. The first polypeptide comprises the amino acid sequence of SEQ ID NO: 41 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 42, or (xx) said first polypeptide. Contains the amino acid sequence of SEQ ID NO: 43 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 44, or (xxi) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 45 and said second. Whether the polypeptide of SEQ ID NO: 46 comprises the amino acid sequence of SEQ ID NO: 46, or (xxiii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 47 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 48. , Or (xxiii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 49 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 50, or (xxiv) the first polypeptide comprises the sequence. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 51 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 52, or (xxv) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 53 and said the second poly. The peptide comprises the amino acid sequence of SEQ ID NO: 54, or (xxvi) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 55 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 56, or The first polypeptide comprises the amino acid sequence of SEQ ID NO: 57 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 58, or (xxviii) the first polypeptide comprises SEQ ID NO: 59. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 60, or (xxx) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 61 and the second polypeptide comprises. The amino acid sequence of SEQ ID NO: 62 is contained, or (xxx) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 63, and the second polypeptide contains the amino acid sequence of SEQ ID NO: 64. Or (xxxi) said first polypeptide comprises the amino acid sequence of SEQ ID NO: 65 and said second polypeptide comprises the amino acid sequence of SEQ ID NO: 66, or (xxxxi) said said first poly. The peptide comprises the amino acid sequence of SEQ ID NO: 67, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 68.

In some embodiments, the compound comprises two said first polypeptide and two said second polypeptide, where the two first polypeptides are associated together by at least one disulfide bond. To do.

In some embodiments, the compound comprises two said first polypeptide and two said said second polypeptide, further here one hinge of the first polypeptide, CH2, and CH3. Associates with the other hinge, CH2, and CH3 of the first polypeptide to form a tetravalent molecule. In some embodiments, the compound comprises two said first polypeptide and two said said second polypeptide, each comprising CH1, hinge, CH2, and CH3. , Each of the second polypeptides comprises CL, wherein one hinge, CH2, and CH3 of the first polypeptide is here with the other hinge, CH2, and CH3 of the first polypeptide. Associate, CH1 of each of the first polypeptides associates with one CL of the second polypeptide to associate with a tetravalent molecule (eg, a monomer, monomer described in the Examples section). Antibodies) (eg, compounds E and V). In some embodiments, the compound comprises two said first polypeptide and two said said second polypeptide, each of the first polypeptide having a third linker, hinge, CH2, and the like. And CH3, each of the second polypeptides comprising a fourth linker, wherein one hinge, CH2, and CH3 of the first polypeptide is the other of the first polypeptide. The third linker of each of the first polypeptide associates with the hinge, CH2, and CH3, and the third linker of each of the first polypeptide associates with the fourth linker of one of the second polypeptide, and is a tetravalent molecule (eg, Example). The monomers, monomeric antibodies described in the section of) (eg, compounds U and T) are formed.

Another aspect of the disclosure relates to a first compound that competes with a second compound for binding to IL-23A and BAFF, said first compound comprising a third polypeptide and a fourth polypeptide. , In this compound
(A) The third polypeptide is
(I) Light chain variable domain (VL1) of the first immunoglobulin specific for the first target protein;
(Ii) Heavy chain variable domain (VH2) of the second immunoglobulin specific for the second target protein; and (iii) Hinge region, heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3). ) Including
(B) The fourth polypeptide is
(I) Light chain variable domain (VL2) of the second immunoglobulin specific for the second target protein;
(Ii) Containing a heavy chain variable domain (VH1) of a first immunoglobulin specific for the first target protein.
Furthermore, in this compound,
(I) The VL1 and VH1 associate to form a binding site that binds to the first target protein.
(Ii) The VL2 and VH2 associate to form a binding site that binds to the second target protein.
(Iii) The first target protein is BAFF and the second target protein is IL-23A, or the first target protein is IL-23A and the second target protein is BAFF. And
Further here, the second compound comprises a first polypeptide and a second polypeptide.
(I) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 5, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 6, or (ii) the first polypeptide comprises SEQ ID NO: 7. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 8 or (iii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 9 and the second polypeptide comprises. Either the amino acid sequence of SEQ ID NO: 10 is contained, or (iv) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 11 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 12 or (v). ) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 13, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 14, or (vi) the first polypeptide comprises the amino acid of SEQ ID NO: 15. The second polypeptide comprises a sequence and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 16 or (vii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 17 and the second polypeptide has the SEQ ID NO: Eighteen amino acid sequences are included, or (viii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 19, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 20 or (ix) said. The first polypeptide comprises the amino acid sequence of SEQ ID NO: 21, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 22, or (x) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 23. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 24, or (xi) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 25 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 26. The first polypeptide comprises the amino acid sequence, or (xii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 27, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 28, or (xiii) said first. The polypeptide comprises the amino acid sequence of SEQ ID NO: 29 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 30 or (xiv) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 31. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 32, or (xv) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 33 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 34. Or (xvi) said first The polypeptide comprises the amino acid sequence of SEQ ID NO: 35 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 36, or (xvii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 37 and said. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 38, or (xviii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 39 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 40. The first polypeptide comprises the amino acid sequence of SEQ ID NO: 41 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 42, or (xx) said first polypeptide. Contains the amino acid sequence of SEQ ID NO: 43 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 44, or (xxi) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 45 and said second. Whether the polypeptide of SEQ ID NO: 46 comprises the amino acid sequence of SEQ ID NO: 46, or (xxiii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 47 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 48. , Or (xxiii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 49 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 50, or (xxiv) the first polypeptide comprises the sequence. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 51 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 52, or (xxv) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 53 and said the second poly. The peptide comprises the amino acid sequence of SEQ ID NO: 54, or (xxvi) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 55 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 56, or The first polypeptide comprises the amino acid sequence of SEQ ID NO: 57 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 58, or (xxviii) the first polypeptide comprises SEQ ID NO: 59. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 60, or (xxx) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 61 and the second polypeptide comprises. The amino acid sequence of SEQ ID NO: 62 is contained, or (xxx) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 63, and the second polypeptide contains the amino acid sequence of SEQ ID NO: 64. Or (xxxi) said first polypeptide comprises the amino acid sequence of SEQ ID NO: 65 and said second polypeptide comprises the amino acid sequence of SEQ ID NO: 66, or (xxxxi) said said first poly. The peptide comprises the amino acid sequence of SEQ ID NO: 67, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 68.

Another aspect of the disclosure relates to a compound comprising two first polypeptides and two second polypeptides, in which the two first polypeptides are associated together by at least one disulfide bond. And each of the first polypeptides associates with one of the second polypeptides by at least one disulfide bond.
Here, each of the first polypeptides is
(I) Light chain variable domain (VL1) of the first immunoglobulin specific for the first target protein;
(Ii) Heavy chain variable domain (VH2) of the second immunoglobulin specific for the second target protein;
(Iii) Includes heavy chain constant region 1 (CH1), hinge region, heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3).
Each of the second polypeptides
(I) Light chain variable domain (VL2) of the second immunoglobulin specific for the second target protein;
(Ii) Heavy chain variable domain (VH1) of the first immunoglobulin specific for the first target protein;
(Iii) Containing the light chain constant region domain (CL)
Here, one hinge, CH2, and CH3 of the first polypeptide associates with the other hinge, CH2, and CH3 of the first polypeptide, and CH1 of each of the first polypeptide is said. It associates with the CL of each of the second polypeptides to form a tetravalent molecule,
In this compound
a) The VL1 and VH1 associate to form a binding site that binds to the first target protein.
b) The VL2 and VH2 associate to form a binding site that binds to the second target protein.
c) The heavy chain constant region 2 (CH2) contains tyrosine at position 252 and threonine at position 254 and glutamic acid at position 256 according to the EU index similar to Kabat.
d) The first target protein is BAFF and the second target protein is IL-23A, or the first target protein is IL-23A and the second target protein is BAFF. Yes,
Furthermore, in this compound,
(I) The VL1 comprises SEQ ID NO: 2, the VH1 comprises SEQ ID NO: 1, the VL2 comprises SEQ ID NO: 4, the VH2 comprises SEQ ID NO: 3, or (ii) the VL1 comprises SEQ ID NO: 4. The VH1 comprises SEQ ID NO: 3, the VL2 comprises SEQ ID NO: 2, the VH2 comprises SEQ ID NO: 1, or (iii) the VL1 comprises SEQ ID NO: 85 and the VH1 comprises SEQ ID NO: 84. The VL2 comprises SEQ ID NO: 4, the VH2 comprises SEQ ID NO: 4, or (iv) the VL1 comprises SEQ ID NO: 4, the VH1 comprises SEQ ID NO: 3, and the VL2 comprises SEQ ID NO: 85. The VH2 comprises SEQ ID NO: 84, or (v) the VL1 comprises SEQ ID NO: 87, the VH1 comprises SEQ ID NO: 86, the VL2 comprises SEQ ID NO: 4, and the VH2 comprises SEQ ID NO: 3. Or (vi) said VL1 comprises SEQ ID NO: 4, said VH1 comprises SEQ ID NO: 3, said VL2 comprises SEQ ID NO: 87, said VH2 comprises SEQ ID NO: 86, or (vii) said. VL1 comprises SEQ ID NO: 89, said VH1 comprises SEQ ID NO: 88, said VL2 comprises SEQ ID NO: 4, said VH2 comprises SEQ ID NO: 3, or (viii) said said VL1 contained SEQ ID NO: 4, said. VH1 comprises SEQ ID NO: 3, said VL2 comprises SEQ ID NO: 89, said VH2 comprises SEQ ID NO: 88, or (ix) said VL1 comprises SEQ ID NO: 91, said VH1 comprises SEQ ID NO: 90, said. VL2 comprises SEQ ID NO: 4, said VH2 comprises SEQ ID NO: 3, or (x) said VL1 comprises SEQ ID NO: 4, said VH1 comprises SEQ ID NO: 3, said VL2 comprises SEQ ID NO: 91, said. VH2 comprises SEQ ID NO: 90.

In some embodiments relating to the above embodiments, each of the first polypeptides further comprises a first linker between the VL1 and the VH2, and each of the second polypeptides is with the VL2. A second linker is further included between the VH1 and the VH1. In some embodiments, the first linker or the second linker comprises the amino acid sequence GGGSGGGG (SEQ ID NO: 69). In some embodiments, the first linker and the second linker comprise the amino acid sequence GGGSGGGG (SEQ ID NO: 69). In some embodiments, each of the first polypeptides further comprises a third linker between the VH2 (or VL1) and CH1, and each of the second polypeptides is said. A fourth linker is further included between VH1 (or said VL2) and said CL. In some embodiments, the third or fourth linker comprises the amino acid sequence LGGGSG (SEQ ID NO: 70). In some embodiments, the third linker and the fourth linker include the amino acid sequence LGGGSG (SEQ ID NO: 70). In some embodiments, the third linker and / or the fourth linker comprises any cysteine residue. In some embodiments, the third linker and / or the fourth linker comprises the amino acid sequence GGCGGGG (SEQ ID NO: 135) or LGGCGGGS (SEQ ID NO: 136). In some embodiments, the heavy chain constant region 2 (CH2) comprises alanine at position 234 and alanine at position 235 according to EU index similar to Kabat. In some embodiments, the amino acid sequence of said hinge region, said heavy chain constant region 2 (CH2), or said heavy chain constant region 3 (CH3) is derived from IgG1 or IgG4. In some embodiments, the hinge region comprises the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 76), the amino acid sequence LEPKSSDKTHTCPPCP (SEQ ID NO: 130), or the amino acid sequence ESKYGPPPCPPCP (SEQ ID NO: 134).

In some embodiments,
(I) Each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 5, and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 6 or (ii) said first polypeptide. Each contains the amino acid sequence of SEQ ID NO: 7, each of the second polypeptide comprises the amino acid sequence of SEQ ID NO: 8, or (iii) each of the first polypeptide comprises the amino acid sequence of SEQ ID NO: 13. Each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 14, or (iv) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 15, said second polypeptide. Each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 16 or (v) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 21 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 22. Or (vi) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 25 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 26, or (vii) said. Each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 29 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 30 or (viii) each of the first polypeptides has a sequence. Each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 34, or (ix) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 37, said. Each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 38, or (x) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 41 and each of the second polypeptides has a sequence. Whether it comprises the amino acid sequence of No. 42, or (xi) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 45 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 46. Alternatively, (xii) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 49 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 50, or (xiii) the first poly. Each of the peptides comprises the amino acid sequence of SEQ ID NO: 53 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 54, or (xiv) said first poly. Each of the peptides comprises the amino acid sequence of SEQ ID NO: 55 and each of the second polypeptide comprises the amino acid sequence of SEQ ID NO: 56, or (xv) each of the first polypeptide comprises the amino acid of SEQ ID NO: 57. The second poly contains a sequence and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 58, or (xvi) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 59. Each of the peptides comprises the amino acid sequence of SEQ ID NO: 60, or (xvii) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 61 and each of the second polypeptides contains the amino acid of SEQ ID NO: 62. Either the sequence is contained or (xviii) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 63 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 64, or (xix). Each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 65 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 66, or (xx) each of the first polypeptides It comprises the amino acid sequence of SEQ ID NO: 67, and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 68.

Another aspect of the disclosure relates to a compound comprising two first polypeptides and two second polypeptides, in which the two first polypeptides are associated together by at least one disulfide bond. And each of the first polypeptides associates with one of the second polypeptides by at least one disulfide bond, and (i) each of the first polypeptides has the amino acid sequence of SEQ ID NO: 5. Each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 6, or (ii) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 7 of the second polypeptide. Each comprises the amino acid sequence of SEQ ID NO: 8, or (iii) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 13 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 14. Either (iv) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 15 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 16 or (v) said said first. Each of the 1 polypeptides comprises the amino acid sequence of SEQ ID NO: 21 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 22 or (vi) each of the first polypeptides has a SEQ ID NO: Each of the second polypeptide comprises 25 amino acid sequences and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 26, or (vii) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 29 and said Each of the two polypeptides comprises the amino acid sequence of SEQ ID NO: 30, or (viii) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 33 and each of the second polypeptides has a SEQ ID NO: Either contains 34 amino acid sequences, or (ix) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 37 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 38. (X) Each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 41 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 42, or (xi) said first polypeptide. Each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 45 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 46, or (xii) said first. Each of the polypeptides comprises the amino acid sequence of SEQ ID NO: 49 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 50, or (xiii) each of the first polypeptides of SEQ ID NO: 53. The second polypeptide comprises an amino acid sequence, each of the second polypeptide comprising the amino acid sequence of SEQ ID NO: 54, or (xiv) each of the first polypeptides comprising the amino acid sequence of SEQ ID NO: 55. Each of the polypeptides comprises the amino acid sequence of SEQ ID NO: 56, or (xv) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 57 and each of the second polypeptides of SEQ ID NO: 58. Either contain an amino acid sequence, or (xvi) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 59, and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 60, or (xvii). ) Each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 61 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 62, or (xviii) each of the first polypeptides. Contains the amino acid sequence of SEQ ID NO: 63 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 64, or (xix) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 65. , Each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 66, or (xx) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 67 and each of the second polypeptides. Contains the amino acid sequence of SEQ ID NO: 68.

Another aspect of the disclosure relates to a compound comprising two first polypeptides and two second polypeptides, in which the two first polypeptides are associated together by at least one disulfide bond. And each of the first polypeptides associates with one of the second polypeptides by at least one disulfide bond.
Here, each of the first polypeptides is
(Iv) Light chain variable domain (VL1) of the first immunoglobulin specific for the first target protein;
(V) Heavy chain variable domain (VH2) of the second immunoglobulin specific for the second target protein;
(Vi) includes a hinge region, a heavy chain constant region 2 (CH2), and a heavy chain constant region 3 (CH3).
Each of the second polypeptides
(I) Light chain variable domain (VL2) of the second immunoglobulin specific for the second target protein;
(Ii) Containing a heavy chain variable domain (VH1) of a first immunoglobulin specific for the first target protein.
Here, one hinge, CH2, and CH3 of the first polypeptide associates with the other hinge, CH2, and CH3 of the first polypeptide.
In this compound
e) The VL1 and VH1 associate to form a binding site that binds to the first target protein.
f) The VL2 and VH2 associate to form a binding site that binds to the second target protein.
g) The heavy chain constant region 2 (CH2) contains tyrosine at position 252 and threonine at position 254 and glutamic acid at position 256 according to the EU index similar to Kabat.
h) The first target protein is BAFF and the second target protein is IL-23A, or the first target protein is IL-23A and the second target protein is BAFF. Yes,
Furthermore, in this compound,
(I) The VL1 comprises SEQ ID NO: 2, the VH1 comprises SEQ ID NO: 1, the VL2 comprises SEQ ID NO: 4, the VH2 comprises SEQ ID NO: 3, or (ii) the VL1 comprises SEQ ID NO: 4. The VH1 comprises SEQ ID NO: 3, the VL2 comprises SEQ ID NO: 2, the VH2 comprises SEQ ID NO: 1, or (iii) the VL1 comprises SEQ ID NO: 85 and the VH1 comprises SEQ ID NO: 84. The VL2 comprises SEQ ID NO: 4, the VH2 comprises SEQ ID NO: 4, or (iv) the VL1 comprises SEQ ID NO: 4, the VH1 comprises SEQ ID NO: 3, and the VL2 comprises SEQ ID NO: 85. The VH2 comprises SEQ ID NO: 84, or (v) the VL1 comprises SEQ ID NO: 87, the VH1 comprises SEQ ID NO: 86, the VL2 comprises SEQ ID NO: 4, and the VH2 comprises SEQ ID NO: 3. Or (vi) said VL1 comprises SEQ ID NO: 4, said VH1 comprises SEQ ID NO: 3, said VL2 comprises SEQ ID NO: 87, said VH2 comprises SEQ ID NO: 86, or (vii) said. VL1 comprises SEQ ID NO: 89, said VH1 comprises SEQ ID NO: 88, said VL2 comprises SEQ ID NO: 4, said VH2 comprises SEQ ID NO: 3, or (viii) said said VL1 contained SEQ ID NO: 4, said. VH1 comprises SEQ ID NO: 3, said VL2 comprises SEQ ID NO: 89, said VH2 comprises SEQ ID NO: 88, or (ix) said VL1 comprises SEQ ID NO: 91, said VH1 comprises SEQ ID NO: 90, said. VL2 comprises SEQ ID NO: 4, said VH2 comprises SEQ ID NO: 3, or (x) said VL1 comprises SEQ ID NO: 4, said VH1 comprises SEQ ID NO: 3, said VL2 comprises SEQ ID NO: 91, said. VH2 comprises SEQ ID NO: 90.

In some embodiments relating to the above embodiments, each of the first polypeptides further comprises a first linker between the VL1 and the VH2, and each of the second polypeptides is with the VL2. A second linker is further included between the VH1 and the VH1. In some embodiments, the first linker or the second linker comprises the amino acid sequence GGGSGGGG (SEQ ID NO: 69). In some embodiments, the first linker and the second linker comprise the amino acid sequence GGGSGGGG (SEQ ID NO: 69). In some embodiments, each of the first polypeptides further comprises a third linker between the VH2 or VL1 and the hinge region, and each of the second polypeptides has the VH1. Alternatively, a fourth linker is further contained at the C-terminal of the VL2. In some embodiments, the third linker of the first polypeptide comprises the amino acid sequence GGCGGGGEVAACEKEVAALEKEVAALEKEVAALEK (SEQ ID NO: 82), and the fourth linker of the second polypeptide is the amino acid sequence GGCGGGKVAACKEKVALKEKALEKVA SEQ ID NO: 83) is included. In another embodiment, the third linker of the first polypeptide comprises the amino acid sequence GGCGGGKVAACKEKVAALKEKVAALKEKVAALKE (SEQ ID NO: 83), and the fourth linker of the second polypeptide is the amino acid sequence GGCGGGGEVAACEKEVAALEKEVAALEKEVA. Includes number 82). In some embodiments, the third linker of the first polypeptide comprises the amino acid sequence GGCGGGGEVAACEKEVAALEKEVAALEKEVAALEK (SEQ ID NO: 82) or the amino acid sequence GGCGGGKVAACKEKVAALKEKVAALKEKALKE (SEQ ID NO: 83). In another embodiment, the fourth linker of the second polypeptide comprises the amino acid sequence GGCGGGGEVAACEKEVAALEKEVAALEKEVAALEK (SEQ ID NO: 82) or the amino acid sequence GGCGGGKVAACKEKVAALKEKVAALKEKALKE (SEQ ID NO: 83). In some embodiments, the third linker comprises the amino acid sequence VEPKSC (SEQ ID NO: 72) or the amino acid sequence FNRGEC (SEQ ID NO: 71). In some embodiments, the fourth linker comprises the amino acid sequence FNRGEC (SEQ ID NO: 71) or the amino acid sequence VEPKSC (SEQ ID NO: 72). In some embodiments, the third linker comprises the amino acid sequence VEPKSC (SEQ ID NO: 72) and the fourth linker comprises the amino acid sequence FNRGEC (SEQ ID NO: 71). In some embodiments, the heavy chain constant region 2 (CH2) comprises alanine at position 234 and alanine at position 235 according to EU index similar to Kabat. In some embodiments, the amino acid sequence of said hinge region, said heavy chain constant region 2 (CH2), or said heavy chain constant region 3 (CH3) is derived from IgG1 or IgG4. In some embodiments, the hinge region comprises the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 76), the amino acid sequence LEPKSSDKTHTCPPCP (SEQ ID NO: 130), or the amino acid sequence ESKYGPPPCPPCP (SEQ ID NO: 134). In some embodiments, (i) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 9, and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 10 or (ii). ) Each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 11 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 12 or (iii) each of the first polypeptides. Contains the amino acid sequence of SEQ ID NO: 17, each of the second polypeptide comprises the amino acid sequence of SEQ ID NO: 18, or (iv) each of the first polypeptide comprises the amino acid sequence of SEQ ID NO: 19. , Each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 20, or (v) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 23 and each of the second polypeptides. Contains the amino acid sequence of SEQ ID NO: 24, or (vi) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 27 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 28. Or (vii) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 31, and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 32, or (viii) said first. Each of the polypeptides of SEQ ID NO: 35 comprises the amino acid sequence of SEQ ID NO: 35, each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 36, or (ix) each of the first polypeptides of SEQ ID NO: 39. Each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 40, or (x) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 43 and said second. Each of the polypeptides of SEQ ID NO: 44 comprises the amino acid sequence of SEQ ID NO: 44, or (xi) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 47 and each of the second polypeptides comprises SEQ ID NO: 48. Each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 51 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 52.

Yet another aspect of the disclosure relates to a pharmaceutical composition comprising the compounds described herein, such as the compounds described above.

Another aspect of the present disclosure relates to a method of treating an autoimmune disease or an inflammatory disease, which comprises administering to a subject a compound described herein, such as the compound described above, or a pharmaceutical composition comprising said compound.

Yet another aspect of the disclosure relates to compounds described herein, such as those described above, for medical use. In some embodiments, the use is the treatment of an autoimmune or inflammatory disease.

Another aspect of the disclosure relates to a pharmaceutical composition comprising a compound described herein, such as the above compounds, used in medicine. In some embodiments, the use is the treatment of an autoimmune or inflammatory disease.

Yet another aspect of the disclosure relates to nucleic acids comprising nucleotide sequences encoding the polypeptides described herein, such as the polypeptides described above. Another aspect of the disclosure relates to a vector containing said nucleic acid. In some embodiments, the vector comprises a promoter operably linked to said nucleic acid. Another aspect of the disclosure relates to cells containing said nucleic acid or said vector.

Another aspect of the disclosure, such as the above-mentioned polypeptide, comprises obtaining a cell described herein, such as the cell described above, and expressing the nucleic acid described herein in the cell. The present invention relates to a method for producing a compound or polypeptide described in the book. In some embodiments, the method further comprises isolating and purifying the polypeptide or compound.

Details of one or more embodiments of the present disclosure are provided in the description below. Other features and advantages of the present disclosure will be apparent from the drawings below and the detailed description of the embodiments and from the appended claims.

The following drawings form part of this specification and are included to illustrate certain aspects of the present disclosure in detail, in conjunction with a detailed description of the particular embodiments presented herein. The present disclosure can be fully understood by reference to one or more of these drawings.

FIG. 5 is a diagram of exemplary compounds specific for BAFF and IL23A. The first polypeptide chain comprises the CH3, CH2, VH ₂ (VH _II ), and VL ₁ (VL _I ) domains. The second polypeptide chain _comprises a VH 1 (VH _I) and _VL 2 _{(VL II)} domains. VL ₁ and VH ₁ are specific for the first target protein (either BAFF or IL23A) and VL ₂ and VH ₂ are for the second target protein (either IL23A or BAFF). Is specific. The upper panel shows each polypeptide chain individually. The lower panel shows a tetravalent compound formed by the association of the CH2 and CH3 domains of one first polypeptide with the CH2 and CH3 domains of another first polypeptide (eg, the section of Examples). The monomers and monomeric antibodies described in (1) are shown. The binding domains for the first and second target proteins are formed by the association of _{VH 1} and VL _1, and by the association of VH ₂ and VL _{2, respectively.} FIG. 5 is a diagram of another exemplary compound specific for BAFF and IL23A. The first polypeptide chain comprises the CH3, CH2, CH1, VH ₂ (VH _II ), and VL ₁ (VL _I ) domains. The second polypeptide _{chain, CL, VH 1 (VH I} ), and a _VL 2 _{(VL II)} domains. VL ₁ and VH ₁ are specific for the first target protein (either BAFF or IL23A) and VL ₂ and VH ₂ are for the second target protein (either IL23A or BAFF). Is specific. The upper panel shows each polypeptide chain individually. The lower panel shows a tetravalent compound formed by the association of the CH2 and CH3 domains of one first polypeptide with the CH2 and CH3 domains of another first polypeptide (eg, the section of Examples). The monomers and monomeric antibodies described in (1) are shown. The binding domains for the first and second target proteins are formed by the association of _{VH 1} and VL _1, and by the association of VH ₂ and VL _{2, respectively.} The compounds are further associated by the interaction between the CL domain and the CH1 domain. Single 1 mg / kg intravenous administration of Compound A (black square), Compound B (white square), Compound C (black triangle), and Compound D (white triangle) in male cynomolgus monkeys according to Example 10. It is a graph which shows the serum concentration (mean ± SD) after. The predicted human serum concentration-time profile of Compound B after subcutaneous administration of 100 mg biweekly as described in Example 11 is shown. The dotted line represents the target C _min (30 nM). The aggregate graph of the monomer percentage with respect to the concentration of compound B described in Example 14 is shown. Compound B is shown to maintain its functional potency against anti-BAFF in vivo. Mice were administered equimolarly with either anti-BAFF or compound B and loaded with a minicircle of human BAFF to induce B cell proliferation. The spleen was collected on day 14, and mouse B220 + B cells were analyzed by flow cytometry as an index of functional blockade of BAFF. Compound B is shown to maintain its functional potency against anti-IL23 in vivo. An evaluation of compound B in an IL23-induced cytokine assay. Mice were administered equimolarly with either anti-IL23 or compound B and human IL23 was loaded twice to induce ear inflammation. Both ears were collected 24 hours after the last injection and analyzed for mouse IL17A and mouse IL22 as indicators of functional blockade of IL23.

This specification describes compounds that bind to both BAFF and IL-23A (also referred to as IL23p19 or IL23A). To date, no approved compound targets both BAFF and IL23A. Limited studies have used biological therapies to simultaneously neutralize two or more important inflammatory mediators, and these studies did not show improved clinical outcome in the assessment of rheumatoid arthritis (RA). .. In addition, such combinations may increase side effects such as risk of infection (eg, Genovese, MC, Cohen, S., Moreland, L., Lium, D., Robbins, S., et. al. (2004). Arth. Rheum. 50, 1412-9; Genovese, MC, Cohen, S., Moreland, L., Lium, D., Robbins, S., et al. (2004). Arth. Rheum. 50, 1412-9; and Weinblatt, M., Schiff, M., Goldman, A. Kremer, J., Lugen, M., et al. (2007). Ann. Rheum. Dis. 66, See 228-34). Moreover, such bispecific compounds have been difficult to design due to problems related to solubility (eg, aggregation) and stability (eg, improper pharmacokinetics).

Surprisingly, some of the BAFF and IL-23A-binding compounds described herein were similar or improved compared to individual antibodies targeting either IL-23A or BAFF. It has been found to have properties. In addition, some compounds were found to have appropriate pharmacokinetics, and showed solubility in a range suitable for the purpose of administration. Moreover, in some embodiments, single doses are advantageous over multiple doses of individual doses, both in terms of side effects of individual therapies and in terms of dose reduction. In addition, in some embodiments, the CMC properties of the compounds showed that some compounds had a high melting temperature (Tm) and low agglomeration. In one aspect, one exemplary compound showed particularly low aggregation at high concentrations. The compounds described herein have one or more advantageous properties compared to standard antibody molecules such as IgG molecules or antigen binding fragments (eg Fab), such as reduced side effects, ease of administration and It is believed to have improved safety, longer half-life, increased binding affinity, or increased inhibitory activity.

Accordingly, aspects of the present disclosure relate to compounds specific for both BAFF and IL-23A, as well as methods of use and production of such compounds. In one aspect, the technique comprises systemic lupus erythematosus (SLE), kidney-related systemic lupus erythematosus / lupus nephritis (LN), ANCA-associated vasculitis (AAV), primary Sjogren's syndrome (pSS), chronic transplant piece-to-host. Autoimmune or inflammatory diseases such as disease (cGVHD), systemic lupus erythematosus (SSc), rheumatoid arthritis (RA), psoriatic arthritis (Ps), tonic spondylitis (AS), and psoriatic arthritis (PA) For compositions with increased efficacy for treatment and prevention. The BAFF / IL23A dual antagonist inhibits both autoantibody / immune complex and IL23-axis mediated terminal organ damage, inducing and maintaining a better clinical response than current standard therapies aimed at treating SLE and LN. Can be achieved. Compared to the simultaneous administration of BAFF antibody and IL-23 antibody to block both pathways at the same time, the BAFF / IL23A dual antagonist can improve medication compliance and relieve pain, for the patient. Convenience is high. The BAFF / IL23A dual antagonist should allow for dose reductions as well as treatment-related costs. In addition, BAFF / IL23A antagonists are also dysregulated, including primary Sjogren's syndrome (pSS), chronic transplant-to-host disease (cGVHD), systemic scleroderma (SSc), and ANCA-associated vasculitis (AAV). It may be beneficial in the treatment of disease groups with B cell / autoantibodies and IL23-mediated tissue damage.

It is difficult to design a dual-target therapeutic molecule with biophysical properties suitable for large-scale production while maintaining the functional efficacy of each component while aggregating the two pharmacological elements. The development of dual target molecules has a number of problems associated with in vitro and in vivo stability, such as inadequate expression, aggregation, shelf life limits, inadequate serum stability, in vivo in vivo imperfections. It has been hampered by appropriate pharmacokinetic properties and the like (Demarest SJ, Glasser SM. (2008). Curr Opin Drug Discov Devel. 11, 675-87).

This specification discloses a method for making a dual target molecule that inhibits the functions of both BAFF and IL23. The dual target molecules of the present technology have advantages and unexpected properties such as high melting temperature (Tm), low cohesiveness at high concentrations, and predicted human PK properties, once every other week or less frequently. Can be administered subcutaneously.

Compounds Aspects of the present disclosure relate to compounds specific for both BAFF and IL23A. An exemplary protein sequence for BAFF and an exemplary protein sequence for IL23A are shown below.

> Sp | Q9Y275 | B cell activating factor BAFF) -TN13B_HUMAN Tumor necrosis factor ligand Superfamily member 13B OS = Homo sapiens GN = TNFSF13B PE = 1 SV = 1
MDDSTEREQSRLTSCLKKREEMKLKECVSILPRKESPSVRSSKDGKLLAATLLLALLSCCLTVVSFYQVAALQGDLASLRAELQGHHAEKLPAGAGAPKAGLEEAPAVTAGLKIFEPPAPGEGNSSQNSRNKRAVQGPEETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRGSALEEKENKILVKETGYFFIYGQVLYTDKTYAMGHLIQRKKVHVFGDELSLVTLFRCIQNMPETLPNNSCYSAGIAKLEEGDELQLAIPRENAQISLDGDVTFFGALKLL (SEQ ID NO: 80)
> NP_057668.1-IL23A [Homo humans]
EmuerujiesuarueibuiemueruerueruerueruPidaburyutieikyujiarueibuiPijijiesuesuPieidaburyutikyushikyukyueruesukyukeierushitierueidaburyuesueieichiPierubuijieichiemudieruaruiijidiiititienudibuiPieichiaikyushijidijishidiPikyujieruarudienuesukyuefushierukyuaruaieichikyujieruaiefuwaiikeieruerujiesudiaiefutijiiPiesueruerupidiesuPibuijikyuerueichieiesueruerujieruesukyueruerukyuPiijieichieichidaburyuitikyukyuaiPiesueruesuPiesukyuPidaburyukyuarueruerueruaruefukeiILRSLQAFVAVAARVFAHGAATLSP (amino acids 1 to 19 predicted signal sequence) (SEQ ID NO: 81)

In some embodiments, the compound comprises a first polypeptide and a second polypeptide. In some embodiments, the first polypeptide is (i) specific for the first target protein, the light chain variable domain (VL1) of the first immunoglobulin, and (ii) specific for the second target protein. Includes a heavy chain variable domain (VH2) of a second immunoglobulin, as well as a (iii) hinge region, a heavy chain constant region 2 (CH2), and a heavy chain constant region 3 (CH3). In some embodiments, the first polypeptide further comprises heavy chain constant region 1 (CH1). In some embodiments, the second polypeptide is (i) specific for the second target protein, the light chain variable domain (VL2) of the second immunoglobulin, and (ii) specific for the first target protein. Contains the heavy chain variable domain (VH1) of a primary immunoglobulin. In some embodiments, the second polypeptide further comprises a light chain constant region (CL).

Understand that the variable domains and constant domains / regions of the first polypeptide may be in any order and the variable domains and constant domains / regions of the second polypeptide (if any) may be in any order. Should. Below, a plurality of exemplary configurations for domains / regions on the first and second polypeptides are shown in order from N-terminus to C-terminus.

Composition of First Polypeptide 1: N-VL1-VH2-Hinge-CH2-CH3-C
Composition of First Polypeptide 2: N-VH2-VL1-Hinge-CH2-CH3-C
Composition of First Polypeptide 3: N-VL1-VH2-CH1-Hinge-CH2-CH3-C
Composition of First Polypeptide 4: N-VH2-VL1-CH1-Hinge-CH2-CH3-C
Composition of the second polypeptide 1: N-VL2-VH1-C
Composition of the second polypeptide 2: N-VH1-VL2-C
Composition of the second polypeptide 3: N-VL2-VH1-CL-C
Composition of the second polypeptide 4: N-VH1-VL2-CL-C

Illustrative configurations of the compounds are shown in FIGS. 1A and 1B.

In some embodiments, the variable regions of the first and second polypeptides associate with each other to form a binding site for the first target protein and a binding site for the second target protein. In some embodiments, the VL1 of the first polypeptide and the VH1 of the second polypeptide associate to form a binding site that binds to the first target protein, and the second polypeptide, VL2, and the second. VH2 of one polypeptide associates to form a binding site that binds to a second target protein. In some embodiments, the first target protein is BAFF and the second target protein is IL23A. In other embodiments, the first target protein is IL23A and the second target protein is BAFF. It should be understood that the terms "first" and "second" are not intended to mean importance to any target protein.

Illustrative sequence combinations for each of VL1, VH1, VL2, and VH2 are shown in Table 1 below and Table 2 of Example 1.

In some embodiments, the compounds are a VL1 sequence containing CDR1, CDR2, and CDR3 of the first light chain, a VH1 sequence containing CDR1, CDR2, and CDR3 of the first heavy chain, and a second light chain. It contains a VL2 sequence containing CDR1, CDR2, and CDR3 of the chain and a VH2 sequence containing CDR1, CDR2, and CDR3 of the second heavy chain. In some embodiments, the CDRs are one or more VL1, VH1, VL2, and VH2 sequences of CDRs listed in Table 1. Illustrative light and heavy chain CDR sequences of the VL1, VH1, VL2, and VH2 sequences listed in Table 1 are shown below:
CDR of SEQ ID NO: 1: GGTFNNNAIN (SEQ ID NO: 92) (CDR1), GIIPMFGTAKYSQNFQG (SEQ ID NO: 93) (CDR2), SRDLLLFPHHALSP (SEQ ID NO: 94) (CDR3)
CDRs of SEQ ID NO: 2: QGDSLRSYYAS (SEQ ID NO: 95) (CDR1), GKNNRPS (SEQ ID NO: 96) (CDR2), SSRDSSGNHWV (SEQ ID NO: 97) (CDR3)
CDR of SEQ ID NO: 3: GYTFTDQTIH (SEQ ID NO: 98) (CDR1), YIYPRDDSPKYNENFKG (SEQ ID NO: 99) (CDR2), PDRSGYAWFIY (SEQ ID NO: 100) (CDR3)
CDR of SEQ ID NO: 4: KASRDVAIAVA (SEQ ID NO: 101) (CDR1), WASTRHT (SEQ ID NO: 102) (CDR2), HQYSSYPFT (SEQ ID NO: 103) (CDR3)
CDR of SEQ ID NO: 84: DHIFSIHWMQ (SEQ ID NO: 104) (CDR1), EIFFGSGTTDYNEKFKG (SEQ ID NO: 105) (CDR2), GAFDY (SEQ ID NO: 106) (CDR3)
CDR of SEQ ID NO: 85: RASQDIGNRLS (SEQ ID NO: 107) (CDR1), ATSSLDS (SEQ ID NO: 108) (CDR2), LQYASSPFT (SEQ ID NO: 109) (CDR3)
CDR of SEQ ID NO: 86: DHIFSIHWMQ (SEQ ID NO: 110) (CDR1), EIFFGSGTTDYNEKFKG (SEQ ID NO: 111) (CDR2), GAFDY (SEQ ID NO: 112) (CDR3)
CDRs of SEQ ID NO: 87: RASQDIGNRLN (SEQ ID NO: 112) (CDR1), ATSSLDS (SEQ ID NO: 113) (CDR2), LQYASSPFT (SEQ ID NO: 114) (CDR3)
CDRs of SEQ ID NO: 88: GYSFSTFFIH (SEQ ID NO: 115) (CDR1), RIDPNSGATKYNEKFES (SEQ ID NO: 116) (CDR2), GEDLLIRTDALDY (SEQ ID NO: 117) (CDR3)
CDRs of SEQ ID NO: 89: KASQNAGIDVA (SEQ ID NO: 118) (CDR1), SKSNRYT (SEQ ID NO: 119) (CDR2), LQYRSYPRT (SEQ ID NO: 120) (CDR3)
CDRs of SEQ ID NO: 90: GYSFSTFFIH (SEQ ID NO: 121) (CDR1), GRIDPNSGATKYNEKFES (SEQ ID NO: 122) (CDR2), GEDLLIRTDALDY (SEQ ID NO: 123) (CDR3)
CDRs of SEQ ID NO: 91: KASQNAGIDVA (SEQ ID NO: 124) (CDR1), SKSNRYT (SEQ ID NO: 125) (CDR2), LQYRSYPRT (SEQ ID NO: 126) (CDR3)

In some embodiments, the compounds are at least 80% (eg, 85%, 90%, 95%, 96%, 97%, per residue unit over the entire sequence), relative to the sequences listed in Table 1. 98% or 99%) Includes VH1, VL1, VH2, and / or VL2 containing sequences that are identical. The "percentage of identity" of the two amino acid sequences is described in Karlin and Altschul Proc. Natl. Acad. Sci. The algorithm of USA 87: 2264-68, 1990 was described in Karlin and Altschul Proc. Natl. Acad. Sci. Modified and used and determined according to USA 90: 5873-77,1993. Such algorithms are described in Altschul, et al. J. Mol. Biol. 215: 403-10, 1990 incorporated into the NBLAST and XBLAST programs (version 2.0). By performing a BLAST protein search with a score of 50 and a word length of 3 in the XBLAST program, an amino acid sequence homologous to the protein molecule of interest can be obtained. If there is a gap between the two sequences, Altschul et al. , Nucleic Acids Res. 25 (17): As described in 3389-3402, 1997, Gapped BLAST can be used. When using BLAST and Gapped BLAST programs, the initialization parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

In some embodiments, the compounds have one or more mutations (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) in the sequences listed in Table 1. Includes VH1, VL1, VH2, and / or VL2 containing the containing sequences. Such mutations can be conservative amino acid substitutions. As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Conservative substitutions of amino acids include, for example, (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S. , T; (f) Q, N; and (g) E, D substitutions made between amino acids within each group.

The amino acid sequences of the compound's hinge region, CH2, and CH3 (possibly CH1 and CL, where the compound comprises such region) can be of any suitable origin, eg, an antibody such as IgG1, IgG2, IgG3, or IgG4. It may be derived from the constant region of. The amino acid sequences of the heavy and light chain constant regions of an antibody are well known in the art and are described, for example, in the IMGT database (www.imgt.org) or www. vbase 2. org / vbstat. php. Any of these sites are incorporated herein by reference. In addition, in some expression systems, the C-terminal lysine residue of the CH3 domain may be removed after translation. Therefore, the C-terminal lysine residue of the CH3 domain is any amino acid residue. Specifically provided by the present invention is a molecule that deletes the C-terminal lysine residue of the CH3 domain. Also specifically included in the present invention are such constructs containing the C-terminal lysine residue of the CH3 domain. In some embodiments, the amino acid sequences of CH2 and CH3 are derived from IgG1 (eg, SEQ ID NO: 75) or IgG4 (eg, SEQ ID NO: 73). In some embodiments, CL comprises the amino acid sequence of Kappa CL or Lambda CL. In some embodiments, the hinge region comprises the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 76). The hinge region of SEQ ID NO: 76 is present, for example, in the polypeptide of SEQ ID NO: 5. In another embodiment, the hinge region comprises the amino acid sequence LEPKSSDKTHTCPPCP (SEQ ID NO: 130). The hinge region of SEQ ID NO: 130 is present, for example, in the polypeptide of SEQ ID NO: 9. The hinge region of SEQ ID NO: 130 also resides at the beginning of the Fc domain of SEQ ID NO: 129. In yet another embodiment, the hinge region comprises the amino acid sequence ESKYGPPPCPPCP (SEQ ID NO: 134). The hinge region of SEQ ID NO: 134 is present, for example, in the polypeptide of SEQ ID NO: 13. The hinge region of SEQ ID NO: 134 also resides at the beginning of the Fc domain of SEQ ID NO: 127.

In some embodiments, the compounds CH2 and / or CH3 (possibly CH1 and CL, where the compound comprises such region) have one or more amino acid substitutions different from wild-type CH2 or CH3, eg, CH2 or CH3 of wild-type IgG1 may contain one or more amino acid substitutions, or CH2 or CH3 of wild-type IgG4 may contain one or more amino acid substitutions (SEQ ID NO: 39 shows exemplary wild-type IgG1). Such substitutions are known in the art (see, eg, US77044497, US7083784, US6821505, US8323962, US6737056, and US7416727).

In some embodiments, CH2 comprises amino acid substitutions at positions 234, 235, 252, 254, and / or 256 according to the EU index similar to Kabat for conventional antibodies (Kabat et al. Sequences). of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, which is incorporated herein by reference in its entirety). It should be understood that all amino acid positions described herein refer to EU index numbers similar to Kabat for conventional antibodies. In some embodiments, CH2 comprises an amino acid substitution at positions 252, 254, and / or 256. In some embodiments, the amino acid at position 252 is tyrosine, phenylalanine, serine, tryptophan, or threonine. In some embodiments, the amino acid at position 254 is threonine. In some embodiments, the amino acid at position 254 is serine, arginine, glutamine, glutamic acid, or aspartic acid. In some embodiments, CH2 contains tyrosine at position 252, threonine at position 254, and glutamic acid at position 256 (referred to herein as the YTE variant). In some embodiments, CH2 comprises an amino acid substitution at position 234 and / or position 235. In some embodiments, CH2 contains alanine at position 234 and alanine at position 235, which is also referred to herein as a KO variant. In some embodiments, CH2 contains tyrosine at position 252, threonine at position 254, glutamic acid at position 256, alanine at position 234, and alanine at position 235, which is also a KO-YTE variant herein. Also called.

In some embodiments, one or more linkers can be used to link the domains / regions in the first and / or second polypeptide into one. For example, the first polypeptide may contain a linker between VL1 and VH2. The first polypeptide further comprises a linker between the VL1 or VH2 (depending on the configuration described above) and the hinge (eg, -VL1-linker-hinge, or -VH2-linker-hinge). If the first polypeptide comprises CH1, for example, the first polypeptide may contain a linker (eg, -VL1-linker-) between VL1 or VH2 and CH1 (depending on the above configuration). CH1 or -VH2-linker-CH1). In another example, the second polypeptide may contain a linker between VL2 and VH1. The second polypeptide may further contain a linker after the C-terminal VL2 or VH1 of the polypeptide chain (eg, -VL2-linker, or -VH1-linker, depending on the above configuration). If the second polypeptide further comprises CL, the second polypeptide may further comprise (depending on the above configuration) a linker between VL2 or VH1 and CL (similarly, eg, -VL2-). Linker-CL, or -VH1-linker-CL). Of course, any number of linkers can be used to link any domain or region to any other domain or region of the first polypeptide, and / or any number of linkers can be used. , Any domain or region can also be linked to any other domain or region of the second polypeptide.

Any suitable linker known in the art is intended for use herein. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker comprises at least two amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. In some embodiments, the peptide linkers are 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 It has an amino acid length of 4, 3, or 2 or less. In some embodiments, the peptide linker is 2-50, 2-40, 2-30, 2-20, 2-10, 2-9, 2-8, 2-7, or 2-6 amino acids long. is there. In some embodiments, the peptide linkers are the amino acid sequences GGGSGGGGG (SEQ ID NO: 69), LGGGSG (SEQ ID NO: 70), FNRGEC (SEQ ID NO: 71), VEPKSC (SEQ ID NO: 72), GGCGGGGEVAACEKEVAALEKEVAALEKEVAALEKAGEKAVALEK (SEQ ID NO: 82). SEQ ID NO: 83) or a combination thereof. In some embodiments, the peptide linker is a plurality of copies of the linker sequence, eg, the sequences GGGSGGGGG (SEQ ID NO: 69), LGGGSG (SEQ ID NO: 70), FNRGEC (SEQ ID NO: 71), VEPKSC (SEQ ID NO: 72) or them. May include multiple copies of the combination of.

In some embodiments, the first and second polypeptides have the following composition:
Composition of First Polypeptide 1: N-VL1-VH2-Hinge-CH2-CH3-C,
Composition of First Polypeptide 2: N-VH2-VL1-Hinge-CH2-CH3-C,
Composition of the second polypeptide 1: N-VL2-VH1-C,
Composition of the second polypeptide 2: It has N-VH1-VL2-C and has
The first linker between VL1 and VH2 of the first polypeptide, or the second linker between VL2 and VH1 of the second polypeptide, comprises the amino acid sequence GGGSGGGG (SEQ ID NO: 69). In some embodiments, the first linker and the second linker comprise the amino acid sequence GGGSGGGG (SEQ ID NO: 69). In some embodiments, the first polypeptide further comprises a third linker between VH2 or VL2 and the hinge region, and the second polypeptide further comprises said VH1 or VL2 (the C-terminus thereof). Includes a fourth linker after. In some embodiments, the third linker of the first polypeptide comprises the amino acid sequence GGCGGGGEVAACEKEVAALEKEVAALEKEVAALEK (SEQ ID NO: 82), and the fourth linker of the second polypeptide is the amino acid sequence GGCGGGKVAACKEKVALKEKALEKVA SEQ ID NO: 83) is included. In another embodiment, the third linker of the first polypeptide comprises the amino acid sequence GGCGGGKVAACKEKVAALKEKVAALKEKVAALKE (SEQ ID NO: 83), and the fourth linker of the second polypeptide is the amino acid sequence GGCGGGGEVAACEKEVAALEKEVAALEKEVA. Includes number 82). In some embodiments, the third linker of the first polypeptide comprises the amino acid sequence GGCGGGGEVAACEKEVAALEKEVAALEKEVAALEK (SEQ ID NO: 82) or the amino acid sequence GGCGGGKVAACKEKVAALKEKVAALKEKALKE (SEQ ID NO: 83). In another embodiment, the fourth linker of the second polypeptide comprises the amino acid sequence GGCGGGGEVAACEKEVAALEKEVAALEKEVAALEK (SEQ ID NO: 82) or the amino acid sequence GGCGGGKVAACKEKVAALKEKVAALKEKALKE (SEQ ID NO: 83). In some embodiments, the third linker comprises the amino acid sequence VEPKSC (SEQ ID NO: 72) or the amino acid sequence FNRGEC (SEQ ID NO: 71). In some embodiments, the fourth linker comprises the amino acid sequence FNRGEC (SEQ ID NO: 71) or the amino acid sequence VEPKSC (SEQ ID NO: 72). In some embodiments, the third linker comprises the amino acid sequence VEPKSC (SEQ ID NO: 72) and the fourth linker comprises the amino acid sequence FNRGEC (SEQ ID NO: 71). The amino acid sequence FNRGEC (SEQ ID NO: 71) is the last 6 amino acid residues of the CL domain (SEQ ID NO: 77), and the amino acid VEPKSC (SEQ ID NO: 72) is the last amino acid of CH1 and the first of the hinge region of SEQ ID NO: 76. Contains 5 amino acid residues (similar to SEQ ID NO: 5).

In some embodiments, the first and second polypeptides have the following composition:
Composition of First Polypeptide 3: N-VL1-VH2-CH1-Hinge-CH2-CH3-C,
Composition of First Polypeptide 4: N-VH2-VL1-CH1-Hinge-CH2-CH3-C,
Composition of the second polypeptide 3: N-VL2-VH1-CL-C,
Composition of Second Polypeptide 4: Having N-VH1-VL2-CL-C
The first linker between the VL1 and the VH2 of the first polypeptide, or the second linker between the VL2 and the VH1 of the second polypeptide, is the amino acid sequence GGGSGGGG (SEQ ID NO: 69). )including. In some embodiments, the first linker and the second linker comprise the amino acid sequence GGGSGGGG (SEQ ID NO: 69). In some embodiments, the first polypeptide further comprises a third linker between the VH2 or VL1 and the CH1, and the second polypeptide further comprises the VH1 or VL2 and the CL. Includes a fourth linker between and. In some embodiments, the third or fourth linker comprises the amino acid sequence LGGGSG (SEQ ID NO: 70). In some embodiments, the third linker and the fourth linker include the amino acid sequence LGGGSG (SEQ ID NO: 70). In some embodiments, the third linker and / or the fourth linker comprises any cysteine residue. In some embodiments, the third linker and / or the fourth linker comprises the amino acid sequence GGCGGGG (SEQ ID NO: 135) or LGGCGGGS (SEQ ID NO: 136).

In some embodiments, the heavy chain constant region 2 (CH2) comprises alanine at position 234 and alanine at position 235 according to EU index numbers similar to Kabat for conventional antibodies.

In some embodiments, the compound comprises two first polypeptides and two second polypeptides. In some embodiments, one hinge, CH2, and CH3 of the first polypeptide associates with the other hinge, CH2, and CH3 of the first polypeptide and is a tetravalent molecule (eg, 2). One first polypeptide is dimerized by association with its respective corresponding hinge, CH2, and CH3 domains, and is specific to the first target protein-specific binding site and the second target protein. Form a tetravalent molecule containing two binding sites), i.e. form the monomeric or monomeric antibody described in the Examples section. If the first polypeptide further comprises the CH1 domain and the second polypeptide further comprises the CL domain, then the CH1 and CL domains are also tetravalent molecules (eg, two first polypeptides each corresponding). Dimerized by association with the hinge, CH2, and CH3 domains, each CH1 of the first polypeptide associates with the CL of one said second polypeptide, and two for the first target protein. It forms a tetravalent molecule containing a binding site and two binding sites for a second target protein), i.e., it may be involved in the formation of the monomers, monomeric antibodies described in the Examples section. In some embodiments, the two first polypeptides are associated together by at least one disulfide bond. In some embodiments, the compound comprises two said first polypeptide and two said said second polypeptide, each of the first polypeptide having a third linker, hinge, CH2, and the like. And CH3, each of the second polypeptides comprising a fourth linker, wherein one hinge, CH2, and CH3 of the first polypeptide is the other of the first polypeptide. The third linker of each of the first polypeptide associates with the hinge, CH2, and CH3, and the third linker of each of the first polypeptide associates with the fourth linker of one of the second polypeptide, and is a tetravalent molecule (eg, Example). The monomers, monomeric antibodies described in the section of) (eg, compounds U and T) are formed.

In some embodiments, the present disclosure relates to a compound comprising two first polypeptides and two second polypeptides, in which the two first polypeptides have at least one disulfide bond. And each of the first polypeptides is associated with one of the second polypeptides by at least one disulfide bond.
Here, each of the first polypeptides is
(I) Light chain variable domain (VL1) of the first immunoglobulin specific for the first target protein;
(Ii) Heavy chain variable domain (VH2) of the second immunoglobulin specific for the second target protein;
(Iii) Includes heavy chain constant region 1 (CH1), hinge region, heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3).
Each of the second polypeptides
(I) Light chain variable domain (VL2) of the second immunoglobulin specific for the second target protein;
(Ii) Heavy chain variable domain (VH1) of the first immunoglobulin specific for the first target protein,
(Iii) Containing the light chain constant region domain (CL)
Here, one hinge, CH2, and CH3 of the first polypeptide associates with the other hinge, CH2, and CH3 of the first polypeptide, and CH1 of each of the first polypeptide is said. It associates with the CL of each of the second polypeptides to form a tetravalent molecule,
In this compound
a) The VL1 and VH1 associate to form a binding site that binds to the first target protein.
b) The VL2 and VH2 associate to form a binding site that binds to the second target protein.
c) The heavy chain constant region 2 (CH2) contains tyrosine at position 252 and threonine at position 254 and glutamic acid at position 256 according to the EU index similar to Kabat.
d) The first target protein is BAFF and the second target protein is IL-23A, or the first target protein is IL-23A and the second target protein is BAFF. Yes,
Furthermore, in this compound,
(I) The VL1 comprises SEQ ID NO: 2, the VH1 comprises SEQ ID NO: 1, the VL2 comprises SEQ ID NO: 4, the VH2 comprises SEQ ID NO: 3, or (ii) the VL1 comprises SEQ ID NO: 4. The VH1 comprises SEQ ID NO: 3, the VL2 comprises SEQ ID NO: 2, the VH2 comprises SEQ ID NO: 1, or (iii) the VL1 comprises SEQ ID NO: 85 and the VH1 comprises SEQ ID NO: 84. The VL2 comprises SEQ ID NO: 4, the VH2 comprises SEQ ID NO: 4, or (iv) the VL1 comprises SEQ ID NO: 4, the VH1 comprises SEQ ID NO: 3, and the VL2 comprises SEQ ID NO: 85. The VH2 comprises SEQ ID NO: 84, or (v) the VL1 comprises SEQ ID NO: 87, the VH1 comprises SEQ ID NO: 86, the VL2 comprises SEQ ID NO: 4, and the VH2 comprises SEQ ID NO: 3. Or (vi) said VL1 comprises SEQ ID NO: 4, said VH1 comprises SEQ ID NO: 3, said VL2 comprises SEQ ID NO: 87, said VH2 comprises SEQ ID NO: 86, or (vii) said. VL1 comprises SEQ ID NO: 89, said VH1 comprises SEQ ID NO: 88, said VL2 comprises SEQ ID NO: 4, said VH2 comprises SEQ ID NO: 3, or (viii) said said VL1 contained SEQ ID NO: 4, said. VH1 comprises SEQ ID NO: 3, said VL2 comprises SEQ ID NO: 89, said VH2 comprises SEQ ID NO: 88, or (ix) said VL1 comprises SEQ ID NO: 91, said VH1 comprises SEQ ID NO: 90, said. VL2 comprises SEQ ID NO: 4, said VH2 comprises SEQ ID NO: 3, or (x) said VL1 comprises SEQ ID NO: 4, said VH1 comprises SEQ ID NO: 3, said VL2 comprises SEQ ID NO: 91, said. VH2 comprises SEQ ID NO: 90.

In some embodiments relating to the above embodiments, each of the first polypeptides further comprises a first linker between the VL1 and the VH2, and each of the second polypeptides is with the VL2. A second linker is further included between the VH1 and the VH1. In some embodiments, the first linker or the second linker comprises the amino acid sequence GGGSGGGG (SEQ ID NO: 69). In some embodiments, the first linker and the second linker comprise the amino acid sequence GGGSGGGG (SEQ ID NO: 69). In some embodiments, each of the first polypeptides further comprises a third linker between the VH2 or VL1 and CH1, and each of the second polypeptides contains the VH1 or A fourth linker is further included between the VL2 and the CL. In some embodiments, the third or fourth linker comprises the amino acid sequence LGGGSG (SEQ ID NO: 70). In some embodiments, the third linker and the fourth linker include the amino acid sequence LGGGSG (SEQ ID NO: 70). In some embodiments, the third linker and / or the fourth linker comprises any cysteine residue. In some embodiments, the third linker and / or the fourth linker comprises the amino acid sequence GGCGGGG (SEQ ID NO: 135) or LGGCGGGS (SEQ ID NO: 136). In some embodiments, the heavy chain constant region 2 (CH2) comprises alanine at position 234 and alanine at position 235 according to EU index similar to Kabat. In some embodiments, the amino acid sequence of said hinge region, said heavy chain constant region 2 (CH2), or said heavy chain constant region 3 (CH3) is derived from IgG1 or IgG4. In some embodiments, the hinge region comprises the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 76), the amino acid sequence LEPKSSDKTHTCPPCP (SEQ ID NO: 130), or the amino acid sequence ESKYGPPPCPPCP (SEQ ID NO: 134). In some embodiments, (i) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 5, and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 6 or (ii). ) Each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 7, each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 8, or (iii) each of the first polypeptides. Contains the amino acid sequence of SEQ ID NO: 13 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 14 or (iv) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 15. , Each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 16, or (v) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 21 and each of the second polypeptides. Contains the amino acid sequence of SEQ ID NO: 22 or (vi) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 25 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 26. Or (vii) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 29 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 30 or (viii) said first. Each of the polypeptides of SEQ ID NO: 33 comprises the amino acid sequence of SEQ ID NO: 33, each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 34, or (ix) each of the first polypeptides of SEQ ID NO: 37. Each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 38, or (x) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 41 and said the second Each of the polypeptides of SEQ ID NO: 42 comprises the amino acid sequence of SEQ ID NO: 42, or (xi) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 45 and each of the second polypeptides comprises SEQ ID NO: 46. , Or (xii) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 49, and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 50, or ( xiii) Each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 53, and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 54, or (Xiv) Each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 55 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 56, or (xv) said first polypeptide. Each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 57 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 58, or (xvi) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 59. Each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 60, or (xvii) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 61, said second polypeptide. Each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 62, or (xviii) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 63, and each of the second polypeptides contains the amino acid sequence of SEQ ID NO: 64. Or (xx) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 65 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 66, or (xx) said. Each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 67, and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 68.

Another aspect of the disclosure relates to a compound comprising two first polypeptides and two second polypeptides, in which the two first polypeptides are associated together by at least one disulfide bond. And each of the first polypeptides associates with one of the second polypeptides by at least one disulfide bond, where (i) each of the first polypeptides is the amino acid of SEQ ID NO: 5. The second poly contains a sequence and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 6, or (ii) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 7. Each of the peptides comprises the amino acid sequence of SEQ ID NO: 8, or (iii) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 13 and each of the second polypeptides contains the amino acid of SEQ ID NO: 14. Either the sequence is contained, or (iv) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 15, and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 16 or (v). Each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 21 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 22 or (vi) each of the first polypeptides Each of the second polypeptide comprises the amino acid sequence of SEQ ID NO: 25 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 26, or (vii) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 29. Each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 30, or (viii) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 33, and each of the second polypeptides Whether it comprises the amino acid sequence of SEQ ID NO: 34, or (ix) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 37 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 38. Or (x) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 41 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 42, or (xi) said first. Each of the polypeptides comprises the amino acid sequence of SEQ ID NO: 45 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 46, or (xii) said. Each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 49 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 50, or (xiii) each of the first polypeptides has a sequence. Each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 54, or (xiv) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 55. Each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 56, or (xv) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 57 and each of the second polypeptides has a sequence. Whether it comprises the amino acid sequence of No. 58, or (xvi) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 59 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 60. Or (xvii) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 61 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 62, or (xvii) said first poly. Each of the peptides comprises the amino acid sequence of SEQ ID NO: 63 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 64, or (xix) each of the first polypeptides contains the amino acid of SEQ ID NO: 65. The second poly contains a sequence and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 66, or (xx) each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 67. Each of the peptides contains the amino acid sequence of SEQ ID NO: 68.

Also contemplated are test compounds that compete with other compounds that compete for binding to the compounds described herein, such as those that compete for binding to BAFF and IL23A. In some embodiments, the test compound is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, with the compounds described herein (in amino acid units over the entire length of the sequence). Or it can have 99% sequence identity. Competitive binding can be determined using any assay known in the art, such as equilibrium binding, ELISA, surface plasmon resonance, or spectroscopy.

In some embodiments, the compounds described herein specifically bind to both BAFF and IL23A. Compounds that "specifically bind" to an antigen or epitope are well known in the art, and methods for determining such specific binding are also well known in the art. When a molecule reacts or associates with a particular target antigen more frequently, rapidly, for a longer period of time, and / or with higher affinity than for another target, the molecule is "specific". It is said to indicate "bonding". When a compound binds with higher affinity, higher binding activity, faster and / or longer duration than it binds to other substances, the compound "specifically" with the target antigen or epitope. Join". For example, a compound that specifically (or preferentially) binds to an antigen (eg, BAFF or IL23A) or its antigenic epitope may bind to this target antigen to another antigen or to another epitope of the same antigen. Is also a compound that has high affinity, high binding activity, and binds more rapidly and / or lasts for a long time. It is also understood by reading this definition that, for example, a compound that specifically binds to a first target antigen may or may not bind specifically or preferentially to a second target antigen. Thus, a "specific bond" or "preferential bond" does not necessarily require (but can include) an exclusive bond. This is not always the case, but in general, when describing a join, it means a preferred join. In some examples, a compound that "specifically binds" to a target antigen or an epitope thereof may not bind to another antigen or another epitope of the same antigen.

In some embodiments, the compounds described herein have appropriate binding affinities for BAFF and IL23 or their antigenic epitopes. As used herein, "binding affinity" refers to the apparent association constant or K _A. K _A is the reciprocal of the dissociation constant _{(K D).} Compounds described herein, to one or both of the target antigen or antigenic epitope, at least ^{10 -5,} 10 ^{-6, 10 -7,} 10 ^{-8, 10 -9,} 10 ^{-10, 10 -11,} 10 ^-12 may have M or less binding affinity _{(K D).} It increased binding affinity corresponds to a decrease in K _D. In some embodiments, the compounds described herein, to one or both of the target antigen or antigenic epitope has at least 10 ^{-11 M} or less for binding affinity (K _D). Compounds than the third antigen is a high binding affinity for the first antigen and the second antigen, than the binding of K _A a third antigen _(or number K _D), the can be represented by the higher of the binding of K _a with one antigen and the second antigen (or number K _D of less) it. In this case, the compound is a first antigen and a second antigen (for example, a first protein having a first three-dimensional structure or a mimic thereof, and a second protein having a first three-dimensional structure or a mimic thereof). ), More specific than a third antigen (eg, the same first or second protein or imitation of the same first or second protein having a second conformation; or a third protein). Differences in binding affinity (eg, for specificity or other comparisons) are at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100. may be 500,1000,10,000 or 10, ^5-fold.

Binding affinity (or binding specificity) can be determined by a variety of methods including equilibrium binding, ELISA, surface plasmon resonance, or spectroscopy (eg, using a fluorescence assay). Exemplary conditions for assessing binding affinity are in HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v / v) Surfactant P20). These techniques can be used to measure the concentration of bound bound protein as a function of the target protein concentration. The concentration of bound binding protein ([binding]) is related to the concentration of the free target protein ([free]) and the concentration of the binding site of the binding protein on the target, where (N) is the binding per target molecule. When it is the number of parts, it is expressed by the following formula:
[Binding] = [N] [Free] / (Kd + [Free])

However, for example, because there is sufficient if if you get a quantitative measure of the affinity method are determined using such as ELISA or FACS analysis, it is not necessarily required for obtaining the K _A accurately. Since this is proportional to K _A, whether a high affinity, for example, by using the comparison of such determining whether 2-fold higher, it and obtain a qualitative measurement of the affinity, or functional assays, Estimates of affinity can be obtained, for example, by activity in in vitro or in vivo assays.

In some embodiments, the compound comprises a first polypeptide and a second polypeptide as defined in Table 2A. In some embodiments, the compound comprises:
(I) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 5, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 6, or (ii) the first polypeptide comprises SEQ ID NO: 7. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 8 or (iii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 9 and the second polypeptide comprises. Either the amino acid sequence of SEQ ID NO: 10 is contained, or (iv) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 11 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 12 or (v). ) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 13, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 14, or (vi) the first polypeptide comprises the amino acid of SEQ ID NO: 15. The second polypeptide comprises a sequence and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 16 or (vii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 17 and the second polypeptide has the SEQ ID NO: Eighteen amino acid sequences are included, or (viii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 19, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 20 or (ix) said. The first polypeptide comprises the amino acid sequence of SEQ ID NO: 21, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 22, or (x) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 23. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 24, or (xi) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 25 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 26. The first polypeptide comprises the amino acid sequence, or (xii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 27, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 28, or (xiii) said first. The polypeptide comprises the amino acid sequence of SEQ ID NO: 29 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 30 or (xiv) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 31. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 32, or (xv) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 33 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 34. Or (xvi) said first The polypeptide comprises the amino acid sequence of SEQ ID NO: 35 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 36, or (xvii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 37 and said. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 38, or (xviii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 39 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 40. The first polypeptide comprises the amino acid sequence of SEQ ID NO: 41 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 42, or (xx) said first polypeptide. Contains the amino acid sequence of SEQ ID NO: 43 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 44, or (xxi) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 45 and said second. Whether the polypeptide of SEQ ID NO: 46 comprises the amino acid sequence of SEQ ID NO: 46, or (xxiii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 47 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 48. , Or (xxiii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 49 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 50, or (xxiv) the first polypeptide comprises the sequence. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 51 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 52, or (xxv) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 53 and said the second poly. The peptide comprises the amino acid sequence of SEQ ID NO: 54, or (xxvi) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 55 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 56, or The first polypeptide comprises the amino acid sequence of SEQ ID NO: 57 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 58, or (xxviii) the first polypeptide comprises SEQ ID NO: 59. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 60, or (xxx) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 61 and the second polypeptide comprises. The amino acid sequence of SEQ ID NO: 62 is contained, or (xxx) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 63, and the second polypeptide contains the amino acid sequence of SEQ ID NO: 64. Or (xxxi) said first polypeptide comprises the amino acid sequence of SEQ ID NO: 65 and said second polypeptide comprises the amino acid sequence of SEQ ID NO: 66, or (xxxxi) said said first poly. The peptide comprises the amino acid sequence of SEQ ID NO: 67, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 68.

Methods for Making Compounds, Nucleic Acids, Vectors, and Cells The aspects of this disclosure also include the compounds described herein or the polypeptides described herein (eg, a first or second poly described herein). A nucleic acid encoding a peptide) is included, but the compound or polypeptide may be encoded together or individually. Any method known in the art can be used to obtain a polynucleotide encoding a compound described herein or a polypeptide described herein, and the nucleotide sequence of the polynucleotide can be determined. it can.

In some embodiments, the nucleic acid is contained within a vector, such as an expression vector. In some embodiments, the vector comprises a promoter operably linked to a nucleic acid.

Various promoters can be used to express the compounds described herein or the polypeptides described herein, including, but not limited to, cytomegalovirus (CMV) intermediate early promoters. , Viral LTRs such as Laus sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Simian virus 40 (SV40) early promoter, E. coli. The coli lac UV5 promoter and the herpes simplex tk virus promoter are included.

Also, adjustable promoters can be used. Such regulatory promoters include E. coli as transcriptional regulators. A colli-derived lac repressor that regulates transcription from a mammalian cell promoter with a lac operator [Brown, M. et al. et al. , Cell, 49: 603-612 (1987)], those using a tetracycline repressor (tetR) [Gossen, M. et al. , And Bujard, H. et al. , Proc. Natl. Acad. Sci. USA 89: 5547-5551 (1992); Yao, F. et al. et al. , Human Gene Therapy, 9: 1939-1950 (1998); Shockelt, P. et al. , Et al. , Proc. Natl. Acad. Sci. USA, 92: 6522-6526 (1995)]. Other systems include FK506 dimer using estradiol, RU486, diphenylmurislerone, or rapamycin, VP16, or p65. Induction systems are available from Invitrogen, Clontech, and Ariad.

Adjustable promoters, including repressors, can be used for operons. In one embodiment, a lac repressor from Escherichia coli is a transcriptional regulator that regulates transcription from a mammalian cell promoter having a lac operator [M. Brown et al. , Cell, 49: 603-612 (1987)]; Gossen and Bujard (1992); [M. Gossen et al. , Natl. Acad. Sci. USA, 89: 5547-5551 (1992)], which can function in combination with the tetracycline repressor (tetR), which, along with the transcriptional activator (VP16), is a tetR mammalian cell transcriptional activator fusion protein, Generate tTa (tetR-VP 16) and generate a tetR-tet operator system that controls gene expression in mammalian cells, along with a mammalian promoter having tetO derived from the human cytomegalovirus (hCMV) major earliest promoter. .. In one embodiment, a tetracycline-inducible switch is used (Yao et al., Human Gene Therapy; Gossen et al., Natl. Acad. Sci. USA, 89: 5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92: 6522-6526 (1995)).

In addition, the vector may include, for example, some or all of the following: selection marker genes such as the neomycin gene for selecting stable or transient transfectants in mammalian cells; high levels of transcription. Enhancer / promoter sequence from the earliest gene of human CMV for; transcription termination and RNA processing signal from SV40 for mRNA stability; SV40 polyoma replication origin and ColE1 for proper episome replication; internal ribosome binding site ( IRES), universal multicloning site; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Vectors and methods suitable for the preparation of vectors containing the transgene are well known and available in the art.

Expression vectors containing nucleic acids can be transferred to host cells by prior art (eg, electroporation, liposome transfection, and calcium phosphate precipitation), and then the transfected cells are cultured by prior art and are described herein. To produce the compounds described in. In some embodiments, expression of the compounds described herein is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

The host cell used to express the compounds described herein or the polypeptides described herein may be bacterial cells such as Escherichia coli, but eukaryotic cells are preferred. In particular, a combination of mammalian cells such as Chinese hamster ovary cells (CHO) with a vector such as a major mid-early gene promoter element derived from human cytomegalovirus is an effective expression system for immunoglobulin (Fooking et al). . (1986) "Powerful And Versatile Enhancer-Promoter Unit For Mammalian Expression Vectors," Gene 45:. 101-106; Cockett et al (1990) "High Level Expression Of Tissue Inhibitor Of Metalloproteinases In Chinese Hamster Ovary Cells Using Glutamine Synthetase Gene Expression, "Biotechnology 8: 662-667).

Various host expression vector systems can be utilized for the expression of the compounds described herein or the polypeptides described herein. Such a host expression system means a medium by which the coding sequence of a compound described herein or a polypeptide described herein can be subsequently produced and subsequently purified, but is suitable. It also means cells capable of expressing the compounds described herein in situ when transformed or transfected with a nucleotide coding sequence. These include microorganisms such as recombinant bacteriopathic DNA, plasmid DNA, or bacteria transformed with a cosmid DNA expression vector (eg, E. coli and B. subtilis) that contain the coding sequences of the compounds described herein. A yeast transformed with a recombinant yeast expression vector containing a sequence encoding a compound described herein (eg, Saccharomyces pichia); a recombinant virus expression vector containing a sequence encoding a compound described herein. Insect cell line infected with (eg, baculovirus): Infected with recombinant virus expression vectors (eg, Califlower Mosaic Virus (CaMV) and Tobacco Mosaic Virus (TMV)) containing sequences encoding the molecular compounds described herein. Plant cell lineaged or transformed with a recombinant plasmid expression vector (eg, Ti plasmid); or mammalian cell lineage (eg, a promoter derived from the mammalian cell genome (eg, metallotionein promoter) or mammalian COS, CHO, BHK, 293,293T, 3T3 cells, lymphoid cells (US Patent No. 5) carrying recombinant expression constructs containing virus-derived promoters (eg, late adenovirus promoters; vaccinia virus 7.5K promoters). , 807, 715), Per C.6 cells (human retinal cells developed by Crucell)), but are not limited thereto.

In the case of a bacterial system, it is advantageous in that a plurality of expression vectors can be selected according to the intended use of the compound to be expressed. For example, when making large quantities of such proteins to produce pharmaceutical compositions of the compounds described herein, vectors that express high levels of readily purified fusion protein products may be desirable. .. As such a vector, the coding sequence can be individually regated to the vector in frame with the lac Z coding region to produce a fusion protein. coli expression vector pUR278 (Ruther et al. (1983) "Easy Identification Of cDNA Cloning," EMBO J.2: 1791-1794), pIN vector (Inouye et al. (1985) "Up-Promoter Escherichia Coli, "Nucleic Acids Res. 13: 3101-3110; Van Heeke et al. (1989)" Expression Of Human Vector Synthesis In Eschebium5: C. , Not limited to these. The pGEX vector can also be used to express a foreign polypeptide as a fusion protein with glutathione-S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to the matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vector is designed to contain a thrombin or factor Xa protease cleavage site so that the cloned target gene product can be released from the GST moiety.

In insects, the Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing foreign genes. The virus grows in Prodenia frugiperda cells. The coding sequence can be individually cloned into a non-essential region of the virus (eg, the polyhedrin gene) and placed under the control of the AcNPV promoter (eg, the polyhedrin promoter).

For mammalian host cells, several viral expression systems can be used. When using an adenovirus as an expression vector, the antibody coding sequence of interest may be ligated with an adenovirus transcription / translation control complex, such as a late promoter and tripartite leader sequence. The chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (eg, region E1 or E3) results in a recombinant virus that is viable in the infected host and capable of expressing an immunoglobulin molecule (eg, Logan et al. (1984) “Adenovirus Tripartite Leader Secequence Enhances Translation Of mRNAs Late After Infection,” Proc. Natl. Acad. Sci. USA 81: 3655-3569). A specific initiation signal may also be required for efficient translation of the inserted antibody coding sequence. Such signals include the ATG start codon and adjacent sequences. In addition, the start codon must match the reading frame of the coding sequence of interest to ensure that all inserts are translated. This exogenous translation control signal and start codon can be of various origins, both natural and synthetic. Expression efficiency can be enhanced by incorporating appropriate transcription enhancer elements, transcription terminators, etc. (see "Expression And Secretion Vectors For Yeast," Methods in Enzymol. 153: 516-544).

In addition, a host cell line that regulates the expression of the inserted sequence, or a host cell line that modifies and processes the gene product in a particular desired manner, can be selected. Such modifications (eg, glycosylation) and processing (eg, cleavage) of the protein product may be important for the function of the protein. For example, in certain embodiments, the compounds described herein can be expressed as a single gene product (eg, as a single polypeptide chain, i.e. as a polyprotein precursor), in which case. , Proteolytic cleavage by natural or recombinant cell mechanisms is required to form the individual polypeptides of the compounds described herein. Accordingly, the present disclosure encodes a polypeptide precursor molecule comprising a polypeptide of a compound described herein and genetically manipulates a nucleic acid sequence containing a coding sequence capable of inducing post-translational cleavage of said polypeptide precursor. Includes. Post-translational cleavage of a polyprotein precursor gives the polypeptide of the compounds described herein. Post-translational cleavage of precursor molecules, including polypeptides of the compounds described herein, is performed in vitro (ie, in a host cell by furin cleavage at a natural or recombinant cell line / mechanism, eg, at the appropriate site. ) In vitro (eg, in a composition containing a protease or peptidase of known activity, and / or in a composition containing conditions or reagents known to promote the desired proteolytic effect). It may be generated by the incubation of the above-mentioned polypeptide chain. Purification and modification of recombinant proteins are well known in the art and therefore the design of a polyprotein precursor may include multiple embodiments that are readily understood by those of skill in the art. Modifications of the precursor molecules described include any protease or peptidase known in the art, such as thrombin or factor Xa (Nagai et al. (1985) "Oxygen Binding Properties Of Human Mutant Hemoglobin Synthesia , "Proc. Nat. Acad. Sci. USA 82: 7252-7255, and Jenny et al. (2003)" A Critical Review Of The Methods For Cleavage Of Fusion Factor X : 1-11, each of which is incorporated herein by reference in its entirety), Escherichia colis-Racie et al. (1995) "Production Of Recombinant Boveine Escherichia , "Biotechnology 13: 982-987, which is incorporated herein by reference in its entirety), Furin, and AcTEV (Parks et al. (1994)" Release Of Precursor And Peptides From Precursor "Anal. Biochem. 216: 413-417, which is incorporated herein by reference in its entirety), and the Escherichia coli virus protease C3 can be used.

Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell or host systems can be selected to ensure accurate modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells with accurate processing of primary transcripts, cell mechanisms for glycosylation and phosphorylation of gene products may be used. Such mammalian host cells include CHO, VERY, BHK, Hela, COS, MDCK, 293,293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030, and Hs578Bst cells. Not limited to these.

Stable expression is preferred in order to produce the recombinant protein in high yield over a long period of time. For example, cell lines that stably express the compounds described herein can be genetically engineered. Instead of using an expression vector containing the origin of replication of the virus, host cells with DNA controlled by appropriate expression control elements (eg, promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers. Can be transformed. After the introduction of the foreign DNA, the genetically engineered cells may be grown in a eutrophic medium for 1 to 2 days and then switched to a selective medium. Selectable markers in recombinant plasmids confer resistance to selection and allow cells to stably integrate the plasmid into their chromosomes and proliferate to form focus. This allows the focus to be cloned and expanded into cell lines. This method may be advantageous when used for genetic engineering of cell lines expressing the compounds described herein. Such genetically engineered cell lines may be particularly useful for screening and evaluation of compounds that interact directly or indirectly with the compounds described herein.

Multiple selection systems can be used and, but are not limited to, for example, herpes simplex virus thymidine kinase (Willer et al. (1977) "Transfer Of Purified Herpes Virus Thymidine Kinase Gene To Celled Mouse Cell" : 223-232), hypoxanthine - guanine phosphoribosyl transferase (. Szybalska et al (1992) "Use Of The HPRT Gene And The HAT Selection Technique In DNA-Mediated Transformation Of Mammalian Cells First Steps Toward Developing Hybridoma Techniques And Gene Therapy, "Bioessays 14: 495-500), and adenine phosphoribosyl transferase (Lowy et al. (1980)" Isolation Of Transfer DNA: Cloning The Hamster virus Gene, "Cell 22: 817-823). They can be used in tk-, hgprt-, or aprt- cells, respectively. In addition, for the following genes, metabolic antagonist resistance can be used as a criterion for selection: dhfr (Wiler et al. (1980) "Transformation Of Mammalian Cells With An Amplifiable Dominant-Acting", which imparts methotrexate resistance. "Proc.Natl.Acad.Sci.USA 77:. 3567-3570; O'Hare et al (1981)" Transformation Of Mouse Fibroblasts To Methotrexate Resistance By A Recombinant Plasmid Expressing A Prokaryotic Dihydrofolate Reductase, "Proc.Natl.Acad. Sci. USA 78: 1527-1531); gpt (Mulligan et al. (1981) "Selection For Animal Cells That Express Strength The Escherichia color Gene Sci. USA 78: 2072-2076); neo (Tolstoshev (1993) "Gene Therapy, Concepts, Current Trials And Future Metabolisms," Ann. 596; Mulligan (1993) "The Basic Science Of Gene Therapy," Science 260: 926-932; and Morgan et al. (1993) "Human Gene Therapy," Ann. Hygro (Santare et al. (1984) "Expression Of P" that imparts resistance to hyglomycin rokaryotic Genes For Hygromycin B And G418 Response As Dominant-Selection Markers In Mouse L Cells, "Gene 30: 147-156). Commonly known methods of recombinant DNA technology in the art that can be used are described in Ausubel et al. (Eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory National; (Eds), 1994, Currant Protocols in Human Genetics, John Wiley & Sons, NY. Colberre-Galapin et al. 1981) "A New Dominant Hybrid Selective Marker For Higher Eukaryotic Cells," J. et al. Mol. Biol. 150: 1-14.

The expression levels of the compounds described herein or the polypeptides described herein can be increased by vector amplification (for an overview, Bebbington and Hentscheel, The use vectors based on genome cloning for the expression of). cloned genes in mammal cells in DNA cloning, Vol. 3 (see Academic Press, New York, 1987). If the markers in the vector system expressing the compounds described herein can be amplified, they are being cultured in host cells. Increasing the level of inhibitor present in increases the number of copies of the marker gene, as the amplified region is associated with the nucleotide sequence of a compound described herein or a polypeptide described herein. , Polypeptide production is also increased (Crowse et al. (1983) "Expression And Application Of Engineered Mouse Dihydropate Vector Minines," Mol. Cell. Biol. 3: 257-2.

The host cell is represented by two expression vectors, a first vector encoding a first polypeptide of a compound described herein and a second polypeptide of a compound described herein. Simultaneous transfection may be performed using the vector of 2. The two vectors may contain the same selectable marker that allows equivalent expression of both polypeptides. Alternatively, a single vector encoding both polypeptides may be used. The coding sequences for the polypeptides of the compounds described herein may include cDNA or genomic DNA.

After recombinant expression of a compound described herein or a polypeptide described herein, with an antibody purification scheme for purifying a polypeptide, protein, or antibody (eg, based on antigen selectivity). Any method known in the art (similar), such as chromatography (eg, ion exchange chromatography, affinity chromatography, especially by affinity for a specific antigen (compound is Fc domain (or part thereof)). After selection of protein A, if included), and size exclusion column chromatography), centrifugation, differential solubility, or purification by any other standard technique for purifying the polypeptide or antibody.

Other aspects of the disclosure relate to cells comprising the nucleic acids described herein or the vectors described herein. The cell may be a prokaryotic cell or a eukaryotic cell. In some embodiments, the cell is a mammalian cell. Illustrative cell types are described herein.

Yet another aspect of the present disclosure is a method of making a compound described herein or a polypeptide described herein (eg, a first polypeptide or a second polypeptide), which is described herein. The present invention relates to a method comprising obtaining the cells described in the above and expressing the nucleic acids described in the present specification in the cells. In some embodiments, the method further comprises isolating and purifying the compounds described herein or the polypeptides described herein.

Therapeutic Methods and Compositions Used in Medicine Other aspects of the disclosure relate to therapeutic methods and compositions used in medicine. Non-limiting examples of compounds used in such methods and compositions include:
(I) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 5, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 6, or (ii) the first polypeptide comprises SEQ ID NO: 7. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 8 or (iii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 9 and the second polypeptide comprises. Either the amino acid sequence of SEQ ID NO: 10 is contained, or (iv) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 11 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 12 or (v). ) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 13, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 14, or (vi) the first polypeptide comprises the amino acid of SEQ ID NO: 15. The second polypeptide comprises a sequence and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 16 or (vii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 17 and the second polypeptide has the SEQ ID NO: Eighteen amino acid sequences are included, or (viii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 19, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 20 or (ix) said. The first polypeptide comprises the amino acid sequence of SEQ ID NO: 21, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 22, or (x) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 23. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 24, or (xi) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 25 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 26. The first polypeptide comprises the amino acid sequence, or (xii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 27, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 28, or (xiii) said first. The polypeptide comprises the amino acid sequence of SEQ ID NO: 29 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 30 or (xiv) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 31. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 32, or (xv) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 33 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 34. Or (xvi) said first The polypeptide comprises the amino acid sequence of SEQ ID NO: 35 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 36, or (xvii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 37 and said. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 38, or (xviii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 39 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 40. The first polypeptide comprises the amino acid sequence of SEQ ID NO: 41 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 42, or (xx) said first polypeptide. Contains the amino acid sequence of SEQ ID NO: 43 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 44, or (xxi) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 45 and said second. Whether the polypeptide of SEQ ID NO: 46 comprises the amino acid sequence of SEQ ID NO: 46, or (xxiii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 47 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 48. , Or (xxiii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 49 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 50, or (xxiv) the first polypeptide comprises the sequence. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 51 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 52, or (xxv) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 53 and said the second poly. The peptide comprises the amino acid sequence of SEQ ID NO: 54, or (xxvi) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 55 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 56, or The first polypeptide comprises the amino acid sequence of SEQ ID NO: 57 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 58, or (xxviii) the first polypeptide comprises SEQ ID NO: 59. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 60, or (xxx) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 61 and the second polypeptide comprises. The amino acid sequence of SEQ ID NO: 62 is contained, or (xxx) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 63, and the second polypeptide contains the amino acid sequence of SEQ ID NO: 64. Or (xxxi) said first polypeptide comprises the amino acid sequence of SEQ ID NO: 65 and said second polypeptide comprises the amino acid sequence of SEQ ID NO: 66, or (xxxxi) said said first poly. The peptide comprises the amino acid sequence of SEQ ID NO: 67, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 68.

In some embodiments, the therapeutic method or use is a method or use of such a method of treating an autoimmune or inflammatory disease. In some embodiments, the method administers a compound described herein or a pharmaceutical composition comprising said compound to a subject, eg, a subject having or at risk of having an autoimmune disease or an inflammatory disease. including.

The subject treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, livestock, competition animals, pets, primates, horses, dogs, cats, mice, and rats. Human subjects in need of treatment can be subjects with or at risk of disease, or subjects with suspected disease. Subjects with the disease can be identified by routine examinations such as physical examinations, clinical examinations, organ function examinations, CT scans, or ultrasound. Subjects suspected of having any of such diseases may exhibit one or more symptoms of the disease. Signs and symptoms of the disease, such as autoimmune and inflammatory diseases, are well known to those of skill in the art. A subject at risk of disease can be a subject having one or more of the risk factors for the disease.

Non-limiting examples of autoimmune diseases include lupus erythematosus (LN) (systemic lupus erythematosus (SLE) associated with the kidneys), systemic lupus erythematosus (SLE), primary Sjogren's syndrome (PSS), Sjogren's disease, and implants. Anti-host disease (GVHD) (eg, chronic transplanted anti-host disease (cGVHD)), systemic lupus erythematosus (SSc), anti-neutrophil cytoplasmic autoimmune (ANCA) -related vasculitis (AAV), rheumatoid arthritis, psoriasis 1, Type 1 diabetes, systemic lupus erythematosus, transplant rejection, autoimmune thyroid disease (Hashimoto's disease), sarcoidosis, sclerosis, granulomatous vasculitis, Crohn's disease, ulcerative colitis, Sjogren's disease, tonic spondylitis , Psoriasis arthritis, lupus erythematosus, nodular polyarteritis, immune-mediated bullskin disease, Bechet syndrome, polysclerosis, good pasture disease, or immune-mediated glomerulonephritis.

Non-limiting examples of inflammatory diseases include rheumatoid arthritis, systemic erythematosus, circular alopecia, tonic dermatomyositis, antiphospholipid syndrome, autoimmune Azison disease, autoimmune hemolytic anemia, autoimmune hepatitis, self. Immune internal ear disease, autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Bechette's disease, bullous vesicles, myocardial disease, celiac disease dermatomyositis, chronic fatigue syndrome (CFIDS), Chronic inflammatory demyelinating polyneuropathy, Scarring tinea, Cold agglutinosis, CREST syndrome, Crohn's disease, Degos's disease, Dermatomyositis, Juvenile dermatomyositis, Discoid lupus, Essential mixture Chryoglobulinemia, primary fibromyositis, Graves' disease, Gillan Valley, Hashimoto dermatomyositis, idiopathic alveolar fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes (Type I), juvenile arteritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, severe myasthenia, asthma vulgaris, malignant anemia, nodular polyarteritis, polychondritis, polyglandular Endocrine syndrome, rheumatic polymyositis, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Reynaud phenomenon, Reiter syndrome, rheumatic fever, sarcoidosis, sclerosis, Schegren syndrome, These include Stiffperson's syndrome, hyperan arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, vasculitis, vasculitis, leukoplakia, and polyangiitis granulomatosis. In some embodiments, the autoimmune or inflammatory disease is Crohn's disease, ankylosing spondylitis, or psoriatic arthritis.

To carry out the methods disclosed herein, an effective amount of a compound or pharmaceutical composition described herein can be administered to a subject in need of treatment (eg, a human). Various delivery systems are known and can be used to administer compounds of the art. Administration methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds of the invention can be administered, for example, by infusion, bolus, or injection, and can be administered with other biologically active agents such as anti-inflammatory agents. Administration can be systemic or topical. In a preferred embodiment, administration is by subcutaneous injection. Such injectable formulations may be, for example, prefilled syringe formulations that can be administered biweekly.

As used herein, "effective amount" refers to the amount of each compound required to exert a therapeutic effect on a subject, either alone or in combination with one or more other compounds. Effective doses, as those skilled in the art are aware, are the individual subject's parameters including the individual condition to be treated, the severity of the condition, age, physical condition, physique, gender and weight, duration of treatment, nature of combination therapy ( Depending on the particular route of administration (if received) and similar factors within the knowledge and experience of the healthcare professional. Such factors are well known to those of skill in the art and can be addressed within the scope of normal experimentation. In general, it is preferred to use the maximum dose of the individual ingredients or combinations thereof, i.e. the maximum safe dose based on sound medical judgment. However, one of ordinary skill in the art should understand that a subject may require a lower dose or tolerated dose for medical, psychological, or virtually any other reason.

Experimental considerations such as half-life generally contribute to dose determination. The use of compounds compatible with the human immune system, such as compounds containing regions from humanized or fully human antibodies, prolongs the half-life of the compound and prevents the host's immune system from attacking the compound. can do. The frequency of administration may be determined and adjusted according to the course of treatment, but generally this is based on the treatment and / or suppression and / or amelioration and / or delay of the disease. Alternatively, a formulation that releases the compound continuously and continuously may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

In some embodiments, the dose is daily, every other day, every 3 days, every 4 days, every 5 days, or every 6 days. In some embodiments, the dosing frequency is once a week, once every two weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks. , Once every 9 weeks, or once every 10 weeks, or once a month, once every two months, once every three months, or once thereafter. The progress of this therapy is easily monitored by prior art and assays. The dosing regimen (including the compounds used) can be changed over time.

In some embodiments, for normal body weight adult subjects, doses ranging from about 0.01 to 1000 mg / kg can be administered. In some embodiments, the dose is 1-200 mg. Individual dosing regimens, ie doses, timings, and repetitions, depend on the individual subject and the medical history of that subject, as well as the characteristics of the compound (eg, the half-life of the compound and other considerations well known in the art). It is determined.

For the purposes of the present disclosure, the appropriate dosage of a compound described herein is the specific compound (or composition thereof) used, the formulation and route of administration, the type and severity of the disease, the administration of the compound. Whether it is for prophylactic or therapeutic purposes is determined by past treatment, the subject's clinical history and responsiveness to the antagonist, and at the discretion of the attending physician. Clinicians usually administer the compound until a dose that achieves the desired result is reached. Administration of one or more compounds is continuous, depending on, for example, the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to the skilled worker. But it may be intermittent. Administration of the compound may be essentially continuous over a preselected period of time, or may be a series of spaced doses, such as before, during, or after the onset of the disease.

As used herein, the term "treating" means disease, symptoms of the disease, or cure, health, alleviate, relieve, change, recovery, ameliorate. , Improve), or the application or administration of a compound or a composition comprising a compound to a subject having a disease, a symptom of a disease, or a predisposition to a disease for the purpose of acting on it.

Relieving the disease involves delaying the onset or progression of the disease or reducing the severity of the disease. Relieving the disease does not necessarily require a cure result. As used herein, "delaying" the onset of a disease means delaying, interfering with, slowing, retarding, stabilizing, and / or postponing the progression of the disease. ) Means to do. This delay can be for varying periods of time, depending on the disease being treated and / or the history of the individual. A method of "delaying" or alleviating the onset of a disease or delaying the onset of a disease causes one or more symptoms of the disease within a given time frame as compared to not using this method. A method of reducing the probability and / or a method of reducing the severity of symptoms within a given time frame. Such comparisons are usually based on clinical trials with multiple subjects that are sufficient to obtain statistically significant results.

"Onset" or "progression" of a disease means the initial symptoms and / or subsequent progression of the disease. The onset of the disease can be detected and evaluated using standard clinical techniques well known in the art. However, onset also refers to progression that may be undetectable. For the purposes of this disclosure, onset or progression refers to the biological course of symptoms. "Onset" includes development, recurrence, and expression. As used herein, "expression" or "development" of a disease includes early onset and / or recurrence.

In some embodiments, the compounds described herein have at least 20% (eg, 30%, 40) activity of one or both of BAFF or IL23A in vivo or in vitro in a subject in need of treatment. %, 50%, 60%, 70%, 80%, 90%, or more) administered in an amount sufficient to inhibit. Methods for determining the inhibitory capacity of a compound are known in the art. An exemplary BAFF and IL23A inhibition assay is described in the Examples.

Depending on the type of disease to be treated or the site of the disease, the compound or pharmaceutical composition can be administered to the subject using conventional methods known to those skilled in the art of medicine. The composition can also be administered by other conventional routes, such as, for example, oral, parenteral, inhalation spray, topical, rectal, nasal, buccal, vaginal, or implantable reservoir administration. As used herein, the term "parenteral" refers to subcutaneous, intradermal, intravenous, intramuscular, joint, intraarterial, synovial, intrasternal, intrathecal, intralesional, and cranial. Includes injecting or injecting techniques. In addition, it can be administered to a subject via an injectable depot route of administration, such as using 1-month, 3-month, or 6-month depot injection or biodegradable materials and methods.

Pharmaceutical Compositions Yet another aspect of the disclosure relates to pharmaceutical compositions comprising the compounds described herein. Compositions comprising compounds of the art (eg, compounds specific for both BAFF and IL23A) can be administered to subjects who have or are at risk of having an autoimmune or inflammatory disease. The technology further provides the use of compounds of the technology in the manufacture of pharmaceuticals for the treatment of autoimmune or inflammatory diseases. The compounds can be administered alone or in combination with other compositions in the prevention or treatment of autoimmune or inflammatory diseases. Non-limiting examples of compounds of the art used in such pharmaceutical compositions include:
(I) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 5, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 6, or (ii) the first polypeptide comprises SEQ ID NO: 7. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 8 or (iii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 9 and the second polypeptide comprises. Either the amino acid sequence of SEQ ID NO: 10 is contained, or (iv) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 11 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 12 or (v). ) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 13, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 14, or (vi) the first polypeptide comprises the amino acid of SEQ ID NO: 15. The second polypeptide comprises a sequence and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 16 or (vii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 17 and the second polypeptide has the SEQ ID NO: Eighteen amino acid sequences are included, or (viii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 19, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 20 or (ix) said. The first polypeptide comprises the amino acid sequence of SEQ ID NO: 21, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 22, or (x) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 23. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 24, or (xi) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 25 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 26. The first polypeptide comprises the amino acid sequence, or (xii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 27, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 28, or (xiii) said first. The polypeptide comprises the amino acid sequence of SEQ ID NO: 29 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 30 or (xiv) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 31. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 32, or (xv) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 33 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 34. Or (xvi) said first The polypeptide comprises the amino acid sequence of SEQ ID NO: 35 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 36, or (xvii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 37 and said. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 38, or (xviii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 39 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 40. The first polypeptide comprises the amino acid sequence of SEQ ID NO: 41 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 42, or (xx) said first polypeptide. Contains the amino acid sequence of SEQ ID NO: 43 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 44, or (xxi) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 45 and said second. Whether the polypeptide of SEQ ID NO: 46 comprises the amino acid sequence of SEQ ID NO: 46, or (xxiii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 47 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 48. , Or (xxiii) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 49 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 50, or (xxiv) the first polypeptide comprises the sequence. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 51 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 52, or (xxv) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 53 and said the second poly. The peptide comprises the amino acid sequence of SEQ ID NO: 54, or (xxvi) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 55 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 56, or The first polypeptide comprises the amino acid sequence of SEQ ID NO: 57 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 58, or (xxviii) the first polypeptide comprises SEQ ID NO: 59. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 60, or (xxx) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 61 and the second polypeptide comprises. The amino acid sequence of SEQ ID NO: 62 is contained, or (xxx) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 63, and the second polypeptide contains the amino acid sequence of SEQ ID NO: 64. Or (xxxi) said first polypeptide comprises the amino acid sequence of SEQ ID NO: 65 and said second polypeptide comprises the amino acid sequence of SEQ ID NO: 66, or (xxxxi) said said first poly. The peptide comprises the amino acid sequence of SEQ ID NO: 67, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 68.

As used herein, the term "pharmaceutical composition" refers to the formulation of a compound described herein in combination with a pharmaceutically acceptable carrier. The pharmaceutical composition can further include additional agents (eg, for specific delivery, half-life extension, or other therapeutic compounds).

As used herein, the term "pharmaceutically acceptable carrier" refers to a compound from one part of the body (eg, a delivery site) to another (eg, an organ, tissue, or part of the body). Liquid or solid bulking agents, diluents, excipients, manufacturing aids (eg, abundant agents, talcmagnesium, calcium stearate or zinc stearate, or stearic acid), or solvent-encapsulated materials involved in transport or transport, etc. Means a pharmaceutically acceptable material, composition, or vehicle. A pharmaceutically acceptable carrier is one that is "acceptable" in the sense that it is compatible with the other ingredients of the formulation and is not harmful to the tissue of interest (eg, physiological compatibility, sterility, physiology). PH, etc.). Some examples of materials that can function as pharmaceutically acceptable carriers are: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) sodium carboxymethyl cellulose, Cellulose such as methyl cellulose, ethyl cellulose, microcrystalline cellulose, and cellulose acetate and derivatives thereof; (4) powdered tragant; (5) malt; (6) gelatin; (7) magnesium stearate, sodium lauryl sulfate, and talc. Esters; (8) Excipients such as cocoa butter and suppository wax; (9) Oils such as peanut oil, cottonseed oil, Benibana oil, sesame oil, olive oil, corn oil, and soybean oil; (10) Propropylene glycol, etc. Glycols; (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) magnesium hydroxide and hydroxides. Buffers such as aluminum; (15) excipients; (16) pyrogen-free water; (17) isotonic physiological saline; (18) Ringer solution; (19) ethyl alcohol; (20) pH buffer solution; (21) polyester , Polycarbonate, and / or polyanhydrides; (22) fillers such as polypeptides and amino acids; (23) serum components such as serum albumin, HDL, and LDL; (22) C2-C12 alcohols such as ethanol; and ( 23) Other compatible non-toxic substances used in pharmaceutical formulations. Formulations may also contain wetting agents, coloring agents, stripping agents, coating agents, sweetening agents, flavoring agents, flavoring agents, preservatives, and antioxidants. The terms "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein.

In some embodiments, the compounds of the art in the composition are administered by injection, catheter, suppository, or implant, the implant being porous, comprising a membrane or fiber, such as a sialastic membrane. It is a non-porous or gelatinous film. Usually, when the composition is administered, a substance that is not absorbed by the compound of the present technology is used.

In other embodiments, the compounds of the art are delivered in a controlled release system. In one embodiment, a pump may be used (eg, Ranger, 1990, Science 249: 1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14: 201; Buchwald et al., 1980, Surgery 88: 507; Saudek et al., 1989, N. Engl. J. Med. 321: 574). In another embodiment, a polymeric material can be used. (E.g., Medical Applications of Controlled Release (Langer and Wise eds, CRC Press, Boca Raton, Fla, 1974);... Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds, Wiley, New York, 1984) See Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. Also, Levy et al., 1985, Science 228: 190; During et al., 1989, Ann. Neurol. 25: 351; Howard et al., 1989, J. Neurosurg. 71: 105). Other controlled release systems are described, for example, in the above Language.

The compounds of the present technology can be administered as a pharmaceutical composition comprising a therapeutically effective amount of a binding agent and one or more pharmaceutically compatible ingredients.

In conventional embodiments, the pharmaceutical composition is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous or subcutaneous administration to a subject, eg, a human. Usually, the composition administered by injection is a solution of sterile isotonic buffer. If desired, the drug may also include a solubilizer and a local anesthetic such as lignokine to relieve pain at the injection site. Generally, the ingredients are delivered individually or in a unit dosage form, eg, as a lyophilized powder or water-free concentrate in a closed container such as an ampoule or sachet indicating the amount of active agent. To. If the drug is administered by injection, it can be dispensed in an infusion bottle containing pharmaceutical grade sterile water or saline. If the drug is administered by injection, the ingredients can be mixed prior to administration by providing an ampoule of sterile water or saline for injection.

The pharmaceutical composition for systemic administration can be a liquid, such as sterile saline, lactated Ringer's solution, or Hanks solution. In addition, the pharmaceutical composition is in solid form and can be redissolved or suspended immediately prior to use. A lyophilized form is also contemplated.

The pharmaceutical composition can be encapsulated in lipid particles or vesicles such as liposomes or microcrystals, which are also suitable for parenteral administration. The particles can be of any suitable structure, such as single layer or multiple layers, as long as the composition is contained therein. The compound contains a fusion-inducible lipid dioleoylphosphatidylethanolamine (DOPE), a low level (5-10 mol%) of cationic lipid, stabilized by polyethylene glycol (PEG) coating, a "stabilizing plasmid". It can be encapsulated in "lipid particles" (SPLP) (Zhang Y.P. et al., Gene Ther. 1999, 6: 1438-47). N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethyl-ammonium methyl sulfate, ie, positively charged lipids such as "DOTAP" are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. For example, U.S. Pat. Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and See No. 4,921,757.

The pharmaceutical compositions of the present disclosure can be administered or packaged, for example, as a unit dose. When used with respect to the pharmaceutical compositions of the present disclosure, the term "unit dose" refers to a physical individual unit suitable as a unit dose to a subject, where each unit is preferred with the required diluent, ie carrier or vehicle. Contains a predetermined amount of active substance calculated to produce a therapeutic effect.

In some embodiments, the compounds described herein may be conjugated to a therapeutic moiety, such as an anti-inflammatory agent. Techniques for conjugating such therapeutic moieties with polypeptides (including, for example, the Fc domain) are well known. For example, Amon et al. , "Monoclonal Antibodies For Immunotarging Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Real. (Eds.), 1985, pp. 243-56, Alan R.M. Liss, Inc. ); Hellstrom et al. , "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (Eds.), 1987, pp. 623-53, Marcel Dekker, Inc. ); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological Electronic Academy. (Eds.), 1985, pp. 475-506); "Analysis, Results, And Future Protective Of The Therapeutic Use Of Radiolabeled Antibodies In Cancer Substance. (Eds.), 1985, pp. 303-16, Academic Press; and Thorpe et al. (1982) "The Preparation And Cytotoxic Properties Of Antibodies-Toxin Conjugates," Immunol. Rev. , 62: 119-158.

Further, the pharmaceutical composition contains (a) a container containing the compound of the present technology in lyophilized form and (b) a second pharmaceutically acceptable diluent for injection (eg, sterile water). Can be provided as a pharmaceutical kit containing the container of. Pharmaceutically acceptable diluents can be used to reconstitute or dilute the lyophilized compounds of the art. In some cases, such containers (s) may be accompanied by a notice in the form prescribed by governmental authorities that regulate the manufacture, use, or sale of pharmaceutical or biological products, and this notice may be attached. Represents regulatory approval for manufacture, use, or sale for human administration.

In another aspect, a product containing a substance useful in treating the above-mentioned diseases is included. In some embodiments, the product comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container may be made of various materials such as glass and plastic. In some embodiments, the container carries a composition that is effective in treating the diseases described herein and may have a sterile access port. For example, the container can be an IV bag or vial with a stopper that can be pierced by a hypodermic needle. The active agent in the composition is a compound of the present technology. In some embodiments, the label on the surface of the container or the label attached to the container indicates that the composition will be used in the treatment of the disease of choice. The product may further comprise a second container containing a pharmaceutically acceptable buffer such as phosphate buffered saline, Ringer's solution, or dextrose solution. Further may include other materials desirable from a commercial and user standpoint, such as buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

Those skilled in the art will be able to make the best use of this disclosure based on the above description without further explanation. Therefore, the following specific embodiments are to be construed as mere examples and do not limit the rest of the disclosure in any case. All publications cited herein are incorporated as references for the purposes or subjects referred to herein.

Example 1. Construction of Illustrative Compounds Targeting IL23A and BAFF The compounds of the present technology are recombinant methods known in the art (see, eg, PCT Publication WO 2006/113665, WO 2008/157379, and WO 2010/080538, all of which. The content is incorporated herein by reference). Table 2A shows exemplary compounds that bind to both IL23A and BAFF used in each of the examples below. Briefly, plasmids encoding the first and second polypeptides for each compound were transfected together into CHO-S cells using the FreeStyle MAX Reagent (CHO). The cells were cultured for 13-14 days and the compounds produced by the cells were purified using protein A chromatography. Compounds were further purified using size exclusion chromatography.

Control antibodies were also used for comparison purposes. Controls were monoclonal antibodies targeting either BAFF or IL23.

Example 2: Thermal Stability of Compounds Thermal unfolding and aggregation of 2 mg / ml phosphate buffer solution of a compound is performed by an automated capillary DSC (MicroCal, LLC, Boston) at a scanning rate of 60 ° C./time at 20 ° C. Monitored at ~ 110 ° C. The apparent heat capacity was determined by performing two scans with the corresponding buffer to check the thermal history of the instrument, obtaining an instrument baseline for each sample, and subtracting the average of both scans from the subsequent protein thermogram. Obtained. Scans were then normalized and analyzed at Origin 7.0. The excess heat capacity (Cp, ex) obtained by subtracting the pre-transition baseline from each obtained heat capacity thermogram was obtained as a function of temperature. The reported value of transition temperature (Tm) represents the position of the maximum peak value determined by visual inspection of the thermogram obtained from the experiment.

Results The results are shown in Table 3. _{The data show that the transition temperature range of the first peak (T m1} ) of the exemplary compounds targeting IL23A and BAFF is in the range 51.59-71.25 ° C. This result is surprising because all of the exemplary compounds had the same overall structure and contained the same VH and VL gene sequences targeting IL-23A. Compounds with higher transition temperatures are expected to be more stable and have longer shelf lives.

Example 3. Surface plasmon resonance (SPR) affinity of exemplary compounds Test compounds were analyzed by SPR to determine their affinity for BAFF and IL23A.

Materials and Methods SPR experiments were performed on the ProteOn XPR36 device (Bio Rad). The GLM chip was preconditioned by sequentially injecting 0.5% SDS, 50 mM NaOH, and 100 mM HCl in both vertical and horizontal directions at a flow rate of 30 μl / min for 60 seconds.

A 1: 1 ratio EDC (76.7 mg / ml) and sulfo-NHS (21.7 mg / ml) mixture was then injected into the horizontal 6 channels to activate the pre-conditioned GLM chips. Goat anti-human IgG (GAHA) Fc gamma (Invitrogen) at a concentration of 30 μg / ml in 10 mM, pH 5.0 sodium acetate buffer to form 8000 resonance units on an activated GLM chip with 6 horizontal channels. Fixed. Finally, 1M ethanolamine HCl was placed in 6 horizontal channels to inactivate the chip. The prepared GAHA chip was rotated vertically to capture the test compound over 5 vertical channels, the last channel being used as a column standard.

The captured chips were then rotated horizontally again to join. Combined human IL-23 (Boehringer Ingelheim Pharmaceuticals, Inc.) at five concentrations of 10.0 nM, 5.00 nM, 2.50 nM, 1.25 nM, and 0.625 nM was added to the following running buffer (Bio Rad). ): Phosphate buffered saline (pH 7.4), injected horizontally over the surface of the test compound at a flow rate of 40 μl / min in 0.005% Tween 20 for 10 minutes. It was dissociated for 2 hours. After 10 minutes of association and 2 hours of dissociation, 0.85% phosphoric acid (BioRad) was shortly pulsed (18 seconds) at a flow rate of 100 μl / min in both horizontal and vertical directions to regenerate the GAHA surface. .. The regenerated GAHA was ready for use in another binding cycle. Binding of the compound to cynomolgus monkey IL23, human BAFF, or cynomolgus monkey BAFF was performed in a similar manner, but the titration concentrations at binding to human BAFF or cynomolgus monkey BAFF were 6.25 nM, 3.12 nM, 1.56 nM, 0. It is .78 nM and 0.39 nM.

result:
The results in Table 4 show that both compounds tested can bind to BAFF and IL23 with a dissociation constant (KD) in the picomolar range.

Example 4. Inhibition of human and cynomolgus monkey BAFF trimer-induced NFkB activation in BAFFR-CHO luciferase reporter cells Materials / Methods:
Briefly, human BAFFR CHO NFkB luciferase reporter cells were collected, washed, counted, and assayed medium (X-VIVO15, a serum-free medium (Lonza) of known composition, plus penicillin / streptomycin 1% (v / v). resuspended at 1.6 × 10 ⁶ cells / ml in AM). Recombinant human or cynomolgus monkey BAFF trimers (Boehringer Ingelheim Pharmaceuticals, Inc.) are prepared in AM at a single concentration (52 pM) and in a humidified incubator at 37 ° C., 5% CO2 for 30 minutes, AM alone, or It was pre-incubated with serially titrated test compounds. After preincubation of BAFF and the test compound, a 50 ul mixture (s) were added to 50 ul cells and the test plates were further incubated (as described above) at 37 ° C. for 24 hours. AM (non-stimulated control) or AM-diluted recombinant BAFF trimer (stimulated control) was added to the control sample. After 24 hours of incubation, cell suspensions were treated with 100 ul of STEADY-Glo reagent (Promega) according to the manufacturer's instructions and assayed for luciferase expression. The resulting relative luminescence (RLU) was plotted against the Log 10 nanomolar concentration of the test compound and the IC ₅₀ and IC ₉₀ values were calculated using a 4-parameter logistic model supported by the Excel add-in Xlfit (ID Business Solutions Limited). did. _{The IC 50} and IC ₉₀ values of the test compounds were calculated as described above, and the geometric mean of the entire plurality of experiments was calculated and shown in Tables 5 and 6.

result:
The test compound dose-dependently inhibited human and cynomolgus monkey BAFF trimer-induced NFkB activation in BAFFR-CHO luciferase reporter cells. The results shown in Tables 5 and 6 show that _{the geometric mean values of IC 50} and IC ₉₀ of the test compounds are comparable to or stronger than the control BAFF antagonists.

Example 5. Inhibition of human BAFF trimer-induced NFkB activation in TACI-CHO luciferase reporter cells Materials / Methods:
Briefly, human TACI CHO NFkB luciferase reporter cells were collected, washed, counted, and assay medium (X-VIVO15, a serum-free medium (Lonza) of known composition, plus penicillin / streptomycin 1% (v / v) resuspended at 1.6 × 10 ⁶ cells / ml in AM). Recombinant human BAFF trimers (Boehringer Ingelheim Pharmaceuticals, Inc.) were prepared in AM at a single concentration (222 pM) and titrated in a humidified incubator at 37 ° C., 5% CO2 for 30 minutes, AM alone or sequentially. Pre-incubated with the test compound. After preincubation of BAFF and the test compound, a 50 ul mixture (s) were added to 50 ul cells and the test plates were further incubated (as described above) at 37 ° C. for 24 hours. Recombinant human BAFF trimer (stimulated control) diluted with AM (non-stimulated control) or AM was added to the control sample. After 24 hours of incubation, cell suspensions were treated with 100 ul of STEADY-Glo reagent (Promega) according to the manufacturer's instructions and assayed for luciferase expression. The resulting relative luminescence (RLU) was plotted against the Log 10 nanomolar concentration of the test compound and the IC ₅₀ and IC ₉₀ values were calculated using a 4-parameter logistic model supported by the Excel add-in Xlfit (ID Business Solutions Limited). did. _{The IC 50} and IC ₉₀ values of the test compounds were calculated as described above, and the geometric mean of the entire plurality of experiments was calculated and shown in Table 7.

result:
The test compound dose-dependently inhibited human BAFF trimer-induced NFkB activation in TACI-CHO luciferase reporter cells. The results shown in Table 7 show that _{the geometric mean values of IC 50} and IC ₉₀ of the test compounds are comparable to or stronger than the control BAFF antagonists.

Example 6. Inhibition of BAFF60-mer-induced NFkB activation in BAFFR-CHO luciferase reporter cells Materials / Methods:
Briefly, human BAFFR CHO NFkB luciferase reporter cells were collected, washed, counted, and assayed medium (X-VIVO15, a serum-free medium (Lonza) of known composition, plus penicillin / streptomycin 1% (v / v). resuspended at 1.6 × 10 ⁶ cells / ml in AM). Recombinant human BAFF60 mer (Boehringer Ingelheim Pharmaceuticals, Inc.) was prepared in AM at a single concentration (4.2 pM) and in a humidified incubator at 37 ° C., 5% CO2 for 30 minutes, AM alone or sequentially. It was pre-incubated with the titrated test compound. After preincubation of BAFF and the test compound, a 50 ul mixture (s) were added to 50 ul cells and the test plates were further incubated (as described above) at 37 ° C. for 24 hours. Recombinant human BAFF60mer (stimulation control) diluted with AM (non-stimulation control) or AM was added to the control sample. After 24 hours of incubation, cell suspensions were treated with 100 ul of STEADY-Glo reagent (Promega) according to the manufacturer's instructions and assayed for luciferase expression. The resulting relative luminescence (RLU) was plotted against the Log 10 nanomolar concentration of the test compound and the IC ₅₀ and IC ₉₀ values were calculated using a 4-parameter logistic model supported by the Excel add-in Xlfit (ID Business Solutions Limited). did. _{The IC 50} and IC ₉₀ values of the test compounds were calculated as described above, and the geometric mean of the entire plurality of experiments was calculated and shown in Table 8.

result:
The test compound dose-dependently inhibited human BAFF60-mer-induced NFkB activation in BAFFR-CHO luciferase reporter cells. _{The results shown in Table 8 show that the geometric mean values of IC 50} and IC ₉₀ of test compounds A, B, C, D, O, P, Q, and R were stronger than all three control BAFF antagonists. Is shown.

Example 7: Neutralization of membrane-bound BAFF-induced NFkB activation in BAFFR-CHO luciferase reporter cells Material / Method:
CHO-K1 cells expressing human BAFF were counted and resuspended in standard growth medium at a concentration of ^{2 × 10 6 cells / ml.} To stop cleavage of membrane-bound BAFF, cells were treated with 0.125% paraformaldehyde (Electron Microscopic) and incubated for 1 hour at room temperature. Then washed fixed human BAFF CHO-K1 cells, in a standard growth medium and resuspended at 2 × 10 ⁶ cells / ml, 37 ° C., in 5% CO ₂ and incubated overnight. Immobilized human BAFF CHO-K1 cells were then harvested and concentrated in X-VIVO15 (Lonza), a serum-free assay medium (AM) of known composition containing penicillin / streptomycin 1% (v / v), at a concentration of 3.2. and resuspended at × 10 ⁶ cells / ml.

Harvested human BAFFR CHO NFkB luciferase reporter cells were washed and resuspended at a concentration 1.6 × 10 ⁶ cells / ml in assay medium. Adding fixed human BAFF CHO-K1 cells, prepared in 3.2 × 10 ⁶ cells / ml in AM, after preincubation sequentially titrated test compound and 30 minutes, the human BAFFR CHO NFkB luciferase reporter cells 50ul And incubated at 37 ° C. for an additional 24 hours. Fixed human BAFF CHO-K1 cells (stimulation control) diluted with AM (non-stimulation control) or AM were added to the control reporter cells. After 24 hours of incubation, samples were treated with 100 ul of STEADY-Glo reagent (Promega) and assayed for luciferase expression. Relative luminescence (RLU) was plotted against _{Log 10 nanomolar concentration of the test compound and IC 50} and IC ₉₀ values were calculated using a 4-parameter logistic model supported by Excel add-in Xlfit (ID Business Solutions Limited). .. _{The IC 50} and IC ₉₀ values of the test compounds were calculated as described above, and the geometric mean of the entire plurality of experiments was calculated and shown in Table 9.

Results The test compound dose-dependently inhibited membrane-bound human BAFF-induced NFkB activation in BAFFR-CHO luciferase reporter cells. The results shown in Table 9 show that the IC ₅₀ and IC ₉₀ values of the test compounds were stronger than all three control BAFF antagonists.

Example 8: Inhibition of human IL-23 activity in lymphoblast B cells (DB cells) using STAT3-luciferase reporter Material / Method:
Lymphoblast B cells (DB cells; ATCC Catalog No. CRL-2289) were stably transduced with the lentivirus STAT-3 / luciferase reporter gene (Qiagen). Transduced cells were selected using puromycin (Life Technologies). The complete medium was RPMI-1640 medium (Life Technologies) supplemented with 10% FBS (Hycrone) and 2 μg / mL puromycin (Life Technologies). The assay medium was RPMI-1640 medium (Life Technologies) supplemented with 10% FBS (Hycrone).

Genetically engineered DB-STAT3 cells were seeded at 20,000 cells / well on a white flat-bottomed 96-well plate containing assay medium at 80 μL / well. Test compound (10-fold) was prepared in a polypropylene round bottom 96-well plate containing assay medium and diluted to a dose range of 1 μg / mL to 10 pg / mL. 10 μL of diluted test molecule or assay medium (for control wells) was added to each of the triple wells. 10 μL of 10-fold human IL-23 (up to 75 ng / mL final concentration per plate) was added to each well. Alternatively, 10 μL of medium was added to the control wells. _{The dose of IL-23 selected for use in the assay represents the EC 60} stimulant dose of human IL-23 for genetically engineered DB cells determined in previous studies. The plates were incubated overnight at 37 ° C. in _{5% CO 2.} ONE-Glo ™ luciferase assay reagent (Promega) was prepared and 100 μL was added to each well and mixed. Luminescence was measured with an Envision plate reader and then plotted against antibody concentration (x-axis) (y-axis). Compound _{IC 50} value was determined by applying the data to a four parameter sigmoidal dose-response function using GraphPad Prism 6 software. The IC ₉₀ value was determined by calculating the data by Find E Planning of GraphPad Prism 6 software. The geometric mean of the entire plurality of experiments was calculated and shown in Table 10.

Results The results showed that the test compound was able to inhibit human IL-23 activity in DB cells by the STAT3-luciferase reporter.

Example 9: Inhibition of human and crab monkey IL-23 activity in mouse spleen Materials and methods Mononuclear cells derived from mouse spleen (female C57BL / 6 younger than 13 weeks; Jackson Laboratories) were isolated, washed and counted. It was resuspended in standard T cell medium (TCM) at 4 × 10 ^{6 cells / ml.} 100 microliters of mIL-2 / splenocyte suspension was added to 96-well microtiter plates. Recombinant human IL-23 (Boehringer Ingelheim Pharmaceuticals, Inc.) or recombinant cynomolgus monkey IL-23 (Boehringer Ingelheim Pharmaceuticals, Inc.) diluted with TCM and incubated with TCM alone or instilled at 2 ° C. .. After preincubation of the test sample and IL-23, 100 ul of the mixture was added to the cells and the test plate was _{incubated with 5% CO 2} humidified air at 37 ° C. for 48 hours. TCM (non-stimulated control) or recombinant IL-23 (stimulated control) diluted with TCM was added to the control sample. After incubation, mouse IL-17 levels were measured from the supernatant using the Quantikine® mouse IL-17 immunoassay according to the manufacturer's instructions (R & D Systems). Interpolated values of mIL-17 pg / ml were measured for each sample and converted to a control percentage (POC). POCs were plotted against test sample concentrations and IC ₉₀ values were calculated using a 4-parameter logistic model enabled by Excel add-in XLfit (Activity Base software, ID Business Solutions, Ltd.). The test compounds were _{analyzed for IC 50} and IC ₉₀ as described above, and the geometric mean of the entire experiment for each test compound was calculated and shown in Table 11.

Results The results showed that the test compound was capable of inhibiting the release of mouse IL-17 in both human and cynomolgus monkey IL23-induced mouse splenocytes.

Example 10. Pharmacokinetics of Compounds in Cynomolgus Monkey Materials and Methods A single intravenous (IV) dose PK test for test compounds A, B, C, and D was performed on male cynomolgus monkeys (n = 2 per molecule). The dose was administered as a slow IV bolus injection of 1 mg / kg. 0.25 hours, 2 hours, and 6 hours after administration on the day of administration, 1 day, 2 days, 3 days, 4 days, 7 days, 10 days, 14 days, 21 days, and 28 days after administration. A whole blood sample was later taken. The serum concentration of the administered molecule was measured by a ligand binding assay using MSD.

Calibration curve and quality control (QC) samples were prepared with 100% pooled cynomolgus monkey serum. Each standard curve consists of 7 non-zero points serially diluted 3-fold from the starting concentration of 512 ng / mL. A blank sample (matrix without analyze) was also included. Five low-range, medium-range, and high-range QC samples were prepared at an starting concentration of 256 ng / mL, then serially diluted 4-fold to 8 ng / mL, and a 2-fold dilution was used to obtain the lowest QC. Prepared at 4 ng / mL. The standard curve and QC sample consisted of two units for each analysis run. The lower and upper limits of quantification were defined as the lowest and highest QC points with reproducible back calculation concentrations where the nominal concentration did not exceed 30 percent. The permissible criterion for standard curve points was 30 percent (%) of the nominal concentration.

To measure the concentration of active drug in serum samples, 0.5 μg / mL biotinylated recombinant human BAFF and in binding buffer (5% BSA in 1x PBS plus 0.05% Tween 20) A master mix was prepared by mixing 0.5 μg / mL of sulfo-labeled goat anti-human IgG detectors. The master mix was added to a 96-well nonbonding light-shielding plate at 50 μL per well. 25 μL of standard and QC material (stock solution diluted 1:20 with binding buffer) was added well-by-well to non-bonding plates containing the master mix. Unknown serum samples were diluted 1:20 with binding buffer and 1: 400 with binding buffer containing 5% serum. A 25 μL diluted sample was added well-by-well to a non-bonding plate containing the master mix. Nonbonding plates were incubated on a plate shaker at room temperature (500 rpm, 1.5 hours). In parallel, MSD streptavidin gold plates were blocked with 150 μL of blocking buffer (5% BSA with 1x PBS plus 0.05% Tween 20) and incubated on a plate shaker at room temperature (500 rpm). , 1.5 hours). After incubation, MSD plates were washed 3 times with 300 μL wash buffer per well (0.05% Tween 20 in 1x PBS). 50 μL of sample from the nonbonding plate was added to the MSD plate and incubated at room temperature (1.5 hours, 600 rpm). After incubation, the plates were washed 3 times with wash buffer, 150 μL of double lead buffer T was added to each well and read immediately with MSD Vector Imager 2400. Standard curves were fitted in a four-parameter logistic manner using MSD Discovery Workbench software. Pharmacokinetic parameters were calculated using a non-compartmental analysis of Phoenix WinNonlin 6.3 (Certara, MD, USA).

result:
The mean (SD) serum concentration vs. time profile for the test compound is shown in FIG. The mean (SD) pharmacokinetic parameters for the test compounds are summarized in Table 12. Serum samples that were confirmed to be positive anti-drug antibodies and showed a sharp decrease in drug concentration over time were excluded from the pharmacokinetic parameter calculation.

Example 11. Prediction of human PK and human dose of an exemplary compound Using an allometry index with a volume of distribution of 1.0 and a clearance of 0.85, using basic Dedrick scaling to obtain the average monkey serum concentration of compound B in humans. Scaled. Predicted human intravenous serum concentration-time profiles were fitted into a linear two-compartment model. The human subcutaneous serum concentration-time profile was predicted by combining parameters from a two-compartment intravenous model with mean subcutaneous absorption rate and bioavailability measured with commercially available therapeutic mAbs. Clearance and terminal phase half-life are predicted to be 0.34 L / d and 9.9 d, respectively, in healthy humans. The predicted human serum concentration-time profile of Compound B after biweekly administration of 100 mg subcutaneous dose is shown in FIG.

The predicted effective dose for humans is 1 mg / kg for biweekly subcutaneous delivery. This dose plan is expected to maintain Cmin ≥ 30 nM (6 μg / mL) with biweekly or less frequent subcutaneous administration. The predicted effective dose may be based on the concentration-PD biomarker response observed for belimumab (Benlysta®), tabalumab, and brishibimod in patients with SLE and RA. These studies have associated maximal inhibition of BAFF-related biomarkers when steady-state Cmin is 30-40 nM. The concentration required for neutralization of IL23 is much lower than that required for neutralization of BAFF and therefore does not affect the Cmin required for the entire dual antagonist.

Example 12. Compound purification method:
Compounds were purified using Mab Select SuRe as an affinity purification step. Elution was performed using sodium acetate buffer pH 3.5. After purification of Mab Select SuRe, the sample was neutralized, added to Poros 50 HS resin and eluted in sodium citrate buffer utilizing a sodium chloride gradient. The monomeric peak elutes with 20 mM sodium citrate and 120 MM NaCl (pH 6.0). After ion exchange chromatography, the samples were consistently> 95% monomeric.

An area under the curve (SV) experiment by ultracentrifugal analysis (AUC) was used to obtain information on sample purity and agglutination status. Samples were centrifuged at 20 ° C. on Optima XL-I (Beckman Coulter, Fullerton, CA) using an An60Ti 4-hole rotor at 40,000 rpm. The corresponding dilution buffer was used as the standard buffer and the precipitation process was monitored by UV absorbance at 280 nm. The time course of the concentration distribution in the ultracentrifugation cell is collected using software running on XL-I and analyzed using the continuous c (S) distribution model of the SEDFIT software (version 14.1). The distribution of the sedimentation coefficient was obtained by. The monomer percentage was calculated based on the integrated peak area.

result:
The results of purification of the compounds are shown in Table 13. The data show that the compound has high purity and homogeneity, which indicates good stability.

Example 13: Mass Spectrometry Profile of Compounds Method:
Undenatured Sample This procedure gave the undenatured mass of a compound or protein. Sample 0.15 ul was injected into an Agilent PoloShell 300SB-C3 column, 5 um (30 x 1.0 mm). The column temperature was 80 ° C. and the flow rate was 150 ul / min. The compound or protein was eluted from the column with a gradient from 10% B at 0 minutes to 85% B at 6 minutes. Mobile phase A is water / acetonitrile / formic acid / ammonium acetate (99/1 / 0.1 / 2 mM), and mobile phase B is n-propanol / acetonitrile / water / formic acid (70/20/10/0. It was 1). The eluate was sent to an Agilent 6224 TOF mass spectrometer and scanned for mass 600-3200. The raw data was deconvolved with the MassHunter program.

Reduced sample By this procedure, the mass of protein or light chain and the mass of heavy chain were obtained. 5 ul of sample was added to 5 uL of a 20: 1 8M guanidine HCL: TCEP mixture and incubated for 15 minutes at room temperature. 0.15 ul of this sample was injected as described above except for the following: compound or protein was eluted from the column with a gradient from 5% B at 0 minutes to 85% B at 6 minutes. The temperature was 60 ° C. and the mass range was 600-2000.

Deglycosylated sample This procedure gave the deglycosylated mass of protein or light and heavy chains. Sample 7.5 ul was added to 3.2 uL of a 20: 1 400 mM ammonium bicarbonate: PNGase F mixture and incubated at 37 ° C. for 3 hours. The sample was then added with 10 ul of a 20: 1 8M guanidine HCL: TCEP mixture and incubated for 15 minutes at room temperature. This sample was injected according to the reduction sample above.

Peptide mapping by mass spectrometry Samples were diluted with denaturing buffer consisting of 6M GdHCl, 250 mM Tris-HCl (pH 7.5), and 10 mM DTT and then incubated at 37 ° C. for 30 minutes. The sample was then alkylated with iodoacetamide and incubated in the dark at room temperature for 30 minutes. The reaction mixture was purified and buffer was replaced with 100 mM Tris-HCL (pH 7.5) using a Sephadex G-25 Superfine cartridge. The sample was then digested with trypsin while incubating at 37 ° C. for 4 hours. The digestion of the reaction mixture was then quenched by the addition of TFA.

The resulting tryptic digest was injected into a Phenomenex Jupiter C18 reverse phase column via a Dionex Ultimate 3000 HPLC autosampler. A gradient solvent system consisting of solvent A: 0.1% formic acid / 99% water / 1% acetonitrile and solvent B: 0.1% formic acid / 5% water / 95% acetonitrile was used. The ratio of solvent B was increased from 0% to 38% over 140 minutes. Chromatographic separation was performed at room temperature at a flow rate of 100 μl / min. The sample storage in the autosampler was 4 ° C. After chromatographic separation, the sample was placed in a Thermo Scientific Orbitrap Fusion mass spectrometer and operated in positive electrospray ionization mode. The methods used included an activated CID utilizing a resolution of 30,000, a minimum signal of 10,000, a separation width of 1.0, and a normalized collision energy of 35.0 V. The S-lens RF level was set to 20%. The data collection type was profile for full MS scan and centroid for CID MS / MS data. Data are collected over a mass range of 250-2000 Da at an acquisition rate of 1 spectrum / sec.

Using Proteome Discover 1.4 (Thermo Scientific), LC-MS and LC-MS / MS raw fragmentation data collected from enzyme digests were analyzed for a given sequence. The identified peptides containing the consensus of N-X-S / T (X is not P) were then analyzed by manually extracting the EIC for the glycosylated peptide. The MS intensity of the glycosylated peptide throughout the EIC was used to estimate the proportion of glycoforms in the total abundance.

result:
The results are shown in Table 14. The data show that the intended amino acid sequence and structure were expressed and recovered without unexpected heterogeneity. The glycosylation pattern is typical of conventional antibodies expressed in CHO cells and does not show any atypical structure.

Example 14: Monomer Content at High Concentrations The purpose of describing this process is to assess the unique properties of compound B by assessing aggregation at high concentrations. A standard buffer of 20 mM sodium citrate, 120 mM NaCl (pH 6) is used to determine the tendency of molecules to aggregate without evaluating the formulation.

Method:
Compound B was gradually concentrated to a maximum concentration at which no precipitation was observed using an Amicon Ultra centrifugal filter (Millipore, Billerica) having a cutoff molecular weight of 50,000 Dalton. The concentrated protein solution was then subjected to analytical SEC analysis to obtain information on sample purity and agglutination status. Chromatography was performed using an Agilent 1200 series HPLC system. The system was run at 1.0 ml / min for 23 minutes. Approximately 30 ug of material was injected into the Tosoh Biosciences TSKgel G3000SWXL column (5 um 250a 7.8 x 30 cm) and the 280 nm results were read. The running buffer used was 50 mM sodium phosphate, 0.2 ML-arginine (pH 6.8).

result:
A summary of analytical SEC data for compound B during the concentration step is shown in Table 15 and FIG. Data showing aggregation and sample purity at high concentrations demonstrate that Compound B does not have a tendency to aggregate at high concentrations.

Example 15: Valence of compound Method:
Using Analytical Membrane-Confined Electrophoresis, the valence of the compound was preliminarily dialyzed against 10 mM acetate, 50 mM KCl (pH 5.0) buffer overnight. In the experimental apparatus, 20 μL of a 1 mg / mL sample was filled in a 2 × 2 × 4 mm3 quartz cuvette whose ends were sealed with a 10 MWCO semi-permeable regenerated cellulose biograde membrane. This membrane traps macromolecules while allowing water and solvent components to pass through. Next, a current of 1 mA was applied to the cuvette to establish an electric field in the long axis direction of the cuvette, and charged macromolecules were moved in the electric field while continuously flowing a fresh buffer solution. Real-time moving density boundaries were detected using a linear photodiode array (LPDA) showing intensity readings separated along the cuvette. Electrophoretic mobility was calculated using the velocity at the concentration boundary, followed by effective valence. Then Stokes radius (obtained from run of sedimentation rate at AUC), counterion radius (0.122 nm for chloride ion), buffer conductance (6.35 mS), and ionic strength (0.05 M), etc. Additional information was used to reveal the basal valences of macromolecules.

result:
Valence data (see Table 16) show the colloidal stability of a compound in solution, i.e., the net interaction between proteins in solution. Compounds with a valence of more than 15 have a strong net repulsive interaction and are likely to be formulated at high concentrations.

Example 16: Whole Blood Stability Method of Compounds Whole blood interference assays were performed on Octet RED96 to detect the effects of non-specific or off-target binding to compounds in the presence of whole blood (WB). Whole blood and 1x kinetic running buffer (1xkb) solutions of the compound were incubated at a temperature of 37 ° C. for 48 hours. Kinetic measurements of the incubated compound samples were performed at 27 ° C. using an Octet RED96 equipped with a streptavidin (SA) biosensor chip (ForteBio, Menlo Park, CA). On-rate / binding signal ratios in buffer and whole blood were recorded. Ratios less than 2 were considered to indicate no interference.

Results The results are shown in Table 17. No whole blood interference was observed with the test compound.

Example 17: Prediction method of in silico immunogenicity The immunogenicity of protein therapeutic agents was described in EpiVax, Inc. Prediction was made in silico using the calculation tool EpiMatrix developed by (Providence, RI). EpiMatrix incorporates predictions of T helper epitopes as well as Treg epitopes, the former eliciting an immune response while the latter being inhibitory. Briefly, protein sequences were first analyzed into overlapping nine-mer peptide frames that have been demonstrated to be the core of Class II HLA binding. The binding potential of the nine-mer peptide to each of the eight common Class II HLA alleles is evaluated based on experimental data or computational predictions. Scores that reflect the binding potential of the nine-mer peptide to each HLA allele can be created, and nine-mer across multiple HLA alleles can be compared, enabling comprehensive immunogenicity prediction. Performed normalization. Finally, the program creates an overall "immunogenicity score", the tReg Adjusted Epx score. This is the likelihood that the compound will elicit an immune response in vivo.

Results The results are shown in Table 18. The overall immunogenicity score of the test compounds is low, and it is predicted that these compounds are unlikely to elicit a strong immune response in vivo.

Example 18: Inhibition of Human BAFF in Primary Human B Cell Proliferation Assay Materials and Methods Normal and healthy blood samples (n = 3 donors) used in this study were purchased from the Biological Specialty Corporation (Philadelphia, PA). The complete medium was IMDM (Life Technologies) supplemented with 10% FBS (Life technologies) + Pen / Strep (Life Technologies). ^{B cell isolation buffer was composed of Mg ++} with 2% FBS + 2 mM EDTA (Life technologies) and sterile DPBS (Life Technologies) with Ca ^{++ free.}

Isolation of human B cells from healthy human whole blood 400 mL of heparinized whole blood was transferred to a 1000 mL sterile polystyrene bottle and an equal volume of DPBS was added. 35 mL of PBS-diluted blood was placed on top of a 15 mL Ficoll-Paque ™ Plus (GE Healthcare) gradient medium prefilled in a 50 mL polystyrene round bottom tube. The tubes were centrifuged at 2000 rpm for 20 minutes at room temperature without brakes. The upper two-thirds of the supernatant was then aspirated and the intermediate gray layer containing peripheral blood mononuclear cells (PBMC) was transferred to a 50 mL conical tube. DPBS (CA ++ and Mg ++ free) + 2% FBS was added up to a volume of 50 mL. The tube was centrifuged at 1200 rpm for 10 minutes. The obtained cell pellet was resuspended in DPBS (CA ++ and Mg ++ free) + 2% FBS (wash buffer), and the centrifugation step (1200 rpm for 10 minutes) was repeated. The cell pellet was resuspended in wash buffer up to 50 mL again. At this point, cell viability and cell concentration were measured.

The tube was then centrifuged at 1200 rpm for 10 minutes and the supernatant was discarded. The cell pellet was resuspended in complete medium, and the cell density was adjusted to 5 × 10 ⁶ cells / mL. The suspended cells were placed in tissue culture flasks 75 cm ^2, and incubated overnight at 5% C0 ₂ incubator (37 ° C.). B cells were then isolated using Dynabeads® Untouched ™ Human B Cell Kits (Life Technologies) according to the manufacturer's protocol. After measuring the cell concentration and viability, the cell concentration was adjusted to be suitable for the downstream procedure.

³ H- Evaluation B cells of B-cell proliferation using thymidine incorporation assay were seeded into 96-well round bottom plates for Falcon tissue culture (1 × 10 ⁵ cells / 100 [mu] L culture medium per well). The test product was then prepared in 4-step concentrations in the range of 0.028-20 nM using complete medium and 4-fold concentrations of B cell stimulants (anti-IgM antibody and human BAFF) (8 μg / mL and 20 ng, respectively). / ML). 50 μL of pre-diluted test product was added to each well (final concentration 0.007-5 nM). Pre-diluted goat anti-human IgM (final concentration 2 μg / mL) and 50 μL hBAFF (final concentration 5 ng / mL or 98 pM) were then added to the corresponding wells (see FIG. 6). _{B cells were cultured in a 5% CO 2} humidified incubator at 37 ° C. for 72 hours. Before 18 hours incubation is terminated by the addition of ³ H- thymidine (Pelkin Elmer) 20μL of 1μCi per well. Using a cell harvester, were transferred ³ H- thymidine incorporation DNA / well in microfiber glass filter plate. At this time, after air-drying the plate for at least 4 hours, 30 μL of scintillation enhancer was added to the wells. The count per minute (cpm) value of each well was measured using a MicroBeta Topcount, and the cpm value (y-axis) with respect to the concentration (x-axis) of the test product was plotted.

Statistical analysis IC ₅₀ and IC ₉₀ were determined by curve fitting the data to a 4-parameter sigmoid dose-response function using GraphPad Prism 6 software. The geometric mean between the three experiments was calculated and shown in Table 19.

Results As the results show, when comparing IC ₅₀ and IC ₉₀ values, compound B was found to be approximately 2-fold more potent than the control antibody 3.

Example 19. B10. Inhibition of B220 + B Cell Number Induced by Overexpression of Human BAFF in RIII Mice Materials and Methods Briefly, B10. RIII female mice (6-8 weeks old, Jackson Laboratory) were randomly divided into 10 groups, 10 animals / group, and tested with citrate buffer (20 mM sodium citrate, 115 mM NaCl, pH 6.0) or equimolar dose. Compounds, 1.3, 0.4 and 0.13 mg / kg vs. 1, 3 and 0.1 mg / kg, respectively, were administered by intraperitoneal injection of 100 μL. Additional controls were untreated naive mice. Immediately after the first day of treatment, mice were administered a single 3 mg dose (1.5 mg / mL) of human BAFF minicircle DNA (System Biosciences) to an empty vector (EV) control group by hydrodynamic injection. Intraperitoneal treatment with either citrate buffer or test compound was repeated every 72 hours on days 3, 6, 9, and 12.

On day 14, mice were anesthetized with isoflurane (Butler Chain) and killed by cervical dislocation. The spleen was resected and the cell suspension was analyzed by flow cytometry for B220 + B cells. The mean number of each treatment group was determined and the significant difference compared to the control was calculated using the Danette multiplex test after one-way ANOVA. The results are shown in FIG.

Results The results showed that treatment with compound B for 14 days could significantly inhibit the proliferation of B220 + B cells induced by the minicircle DNA of human BAFF.

Example 20. Inhibition of IL17A and IL22 release in human IL23-induced mice in C57 / Bl6 mice Materials and methods Briefly, C57BL / 6 female mice (7-10 weeks old, Charles River) were randomly selected in 8 groups, 8 animals / group. Divided into citrate buffer (20 mM sodium citrate, 115 mM NaCl, pH 6.0) or equimolar doses of test compound, 1.3, 0.4 and 0.13 mg / kg vs. 1, 3 and 0.1 mg, respectively. / Kg was administered by intraperitoneal injection of 100 μL.

One hour after administration of the test compound, mice were anesthetized with isoflurane (Butler Chain) and diluted with saline (Invitrogen) 0.1% BSA (Sigma) control or 15 μg / ml (0.3 μg) rhIL23 (in-house). ) Was administered by intradermal injection of 20 μL into both ears. Subcutaneous loading was repeated daily for 2 consecutive days. Twenty-four hours after the second loading, mice were sacrificed to death by cervical dislocation and each ear was resected. Homogenize ear tissue in 1 ml of homogenized buffer (HBSS (Gibco); 0.4% Triton X-100 (Sigma); 1X SigmaFast Protease Inhibitor (Sigma)) using MP Biomedicals Fast-Prep 24 Homogenizer. did. The homogenized sample was centrifuged at 4 ° C. for 10 minutes and the supernatant was collected. The supernatants were assayed for the presence of mouse IL17A and IL22 using the Quantikine® mouse IL-17 and mouse IL-22 immunoassays according to the manufacturer's instructions (R & D Systems). Interpolated values of cytokine pg / ml were measured for each sample. Mean pg / ml levels for each treatment group were determined and significant differences compared to controls were calculated using the Danette multiplex test after one-way ANOVA.

Results The results in FIG. 6 show that treatment with a single intraperitoneal dose of Compound B can significantly inhibit the release of mouse IL17 and IL22 in the skin induced by two consecutive days of endothelial injection of recombinant human IL23. Indicated.

Other Embodiments All the functions disclosed herein may be combined in any combination. Each function disclosed herein can be replaced by an alternative function that serves the same, equivalent, or similar purpose. Therefore, unless otherwise stated, each disclosed feature is merely an example of a general set of equivalent or similar features.

From the above description, those skilled in the art can easily confirm the essential features of the present disclosure, and make various changes and modifications to the present disclosure without departing from the spirit and scope of the present disclosure. The present disclosure can be adapted to a variety of uses and conditions. Therefore, other embodiments are also within the scope of the claims.

Claims (23)

A compound containing a first polypeptide and a second polypeptide.
(A) The first polypeptide is prepared from the N-terminal to the C-terminal in the following directions:
(I) Light chain variable domain (VL1) of the first immunoglobulin specific for the first target protein;
(Ii) First linker;
(Iii) Heavy chain variable domain (VH2) of a second immunoglobulin specific for a second target protein;
(Iv) Third linker;
(V) Heavy chain constant region 1 (CH1);
(Vi) Hinge area;
Heavy chain constant region 2 (CH2); and (viii) heavy chain constant region 3 (CH3) containing tyrosine at position 252 and threonine at position 254 and glutamic acid at position 256 according to EU index similar to (vii) Kabat. ;
Including
(B) The second polypeptide is prepared from the N-terminal to the C-terminal in the following directions:
(I) Light chain variable domain (VL2) of the second immunoglobulin specific for the second target protein;
(Ii) Second linker;
(Iii) Heavy chain variable domain (VH1) of a first immunoglobulin specific for the first target protein;
(Iv) Fourth linker; and (v) Light chain constant region (CL);
Including
Here, in the compound,
a) The VL1 and VH1 associate to form a binding site that binds to the first target protein; and b) the VL2 and VH2 associate to form a binding site that binds to the second target protein. Form;
here,
(I) The first target protein is the p19 subunit (IL-23A) of interleukin 23 and the second target protein is B cell activating factor (BAFF).
(I) The VL1 contains the amino acid sequence of SEQ ID NO: 4, the VH1 contains the amino acid sequence of SEQ ID NO: 3, the VL2 contains the amino acid sequence of SEQ ID NO: 91, and the VH2 contains the amino acid sequence of SEQ ID NO: 90. Is it a waste;
(Ii) The VL1 contains the amino acid sequence of SEQ ID NO: 4, the VH1 contains the amino acid sequence of SEQ ID NO: 3, the VL2 contains the amino acid sequence of SEQ ID NO: 89, and the VH2 contains the amino acid sequence of SEQ ID NO: 88. Or (iii) said VL1 comprises the amino acid sequence of SEQ ID NO: 4, said VH1 contained the amino acid sequence of SEQ ID NO: 3, said VL2 contained the amino acid sequence of SEQ ID NO: 2, and said VH2 contained the amino acid sequence of SEQ ID NO: 1. It contains an amino acid sequence;
Or
(II) The first target protein is BAFF and the second target protein is IL-23A.
The VL1 comprises the amino acid sequence of SEQ ID NO: 2, the VH1 comprises the amino acid sequence of SEQ ID NO: 1, the VL2 comprises the amino acid sequence of SEQ ID NO: 4, and the VH2 comprises the amino acid sequence of SEQ ID NO: 3;
The compound. The compound according to claim 1, wherein the first linker and the second linker include the amino acid sequence of SEQ ID NO: 69. The compound according to claim 1 or 2, wherein the third linker and the fourth linker include the amino acid sequence of SEQ ID NO: 70. The compound according to any one of claims 1 to 3, which comprises one or more of the following characteristics:
(I) The CH2 contains alanine at position 234 and alanine at position 235 according to the EU index similar to Kabat;
(Ii) The amino acid sequences of CH1, the hinge region, CH2, and CH3 are derived from IgG1 or IgG4;
(Iii) The hinge region comprises the amino acid sequence of SEQ ID NO: 76, the amino acid sequence of SEQ ID NO: 130, or the amino acid sequence of SEQ ID NO: 134. The compound according to any one of claims 1 to 3.
(I) The amino acid sequences of the CH1, the hinge region, the CH2, and the CH3 are derived from IgG1, the hinge region contains the amino acid sequence of SEQ ID NO: 76, and the CH2 is the same EU as Kabat. Does the index number contain alanine at position 234 and alanine at position 235; or (ii) the amino acid sequences of CH1, said hinge region, said CH2, and said CH3 are derived from IgG4 and said hinge region. Contains the amino acid sequence of SEQ ID NO: 134;
The compound. The compound according to claim 1.
(I) Does the first polypeptide contain the amino acid sequence of SEQ ID NO: 5 and the second polypeptide contains the amino acid sequence of SEQ ID NO: 6;
(Ii) Does the first polypeptide contain the amino acid sequence of SEQ ID NO: 15 and the second polypeptide contains the amino acid sequence of SEQ ID NO: 16;
(Iii) Does the first polypeptide contain the amino acid sequence of SEQ ID NO: 37 and the second polypeptide contains the amino acid sequence of SEQ ID NO: 38;
(Iv) Does the first polypeptide contain the amino acid sequence of SEQ ID NO: 41 and the second polypeptide contains the amino acid sequence of SEQ ID NO: 42;
(V) Is the first polypeptide comprising the amino acid sequence of SEQ ID NO: 45 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 46; or (vi) the first polypeptide comprises SEQ ID NO: 49. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 50;
The compound. The compound comprises two said first polypeptide and two said said second polypeptide, the two first polypeptides are associated together by at least one disulfide bond, and said said first polypeptide. The compound according to any one of claims 1 to 6, wherein each of the compounds associates with one of the second polypeptides by at least one disulfide bond to form a tetravalent molecule. A compound containing two first polypeptides and two second polypeptides.
The two first polypeptides are associated together by at least one disulfide bond, and each of the first polypeptides is associated with one of the second polypeptides by at least one disulfide bond and is tetravalent. Form molecules, where
(A) Each of the first polypeptides is described below in the direction from the N-terminal to the C-terminal:
(I) VL1 specific for the first target protein;
(Ii) First linker;
(Iii) VH2 specific for the second target protein;
(Iv) Third linker;
(V) CH1;
(Vi) Hinge area;
CH2; and (viii) CH3; containing tyrosine at position 252 and threonine at position 254 and glutamic acid at position 256 according to EU index similar to (vii) Kabat;
Including
(B) Each of the second polypeptides is described below in the direction from the N-terminal to the C-terminal:
(I) VL2 specific for the second target protein;
(Ii) Second linker;
(Iii) VH1 specific for the first target protein;
(Iv) 4th linker; and (v) CL;
Including
Here, in the compound,
a) The VL1 and VH1 associate to form a binding site that binds to the first target protein; and b) the VL2 and VH2 associate to form a binding site that binds to the second target protein. Form;
here,
(I) The first target protein is IL-23A and the second target protein is BAFF, where further.
(I) The VL1 contains the amino acid sequence of SEQ ID NO: 4, the VH1 contains the amino acid sequence of SEQ ID NO: 3, the VL2 contains the amino acid sequence of SEQ ID NO: 91, and the VH2 contains the amino acid sequence of SEQ ID NO: 90. Is it a waste;
(Ii) The VL1 contains the amino acid sequence of SEQ ID NO: 4, the VH1 contains the amino acid sequence of SEQ ID NO: 3, the VL2 contains the amino acid sequence of SEQ ID NO: 89, and the VH2 contains the amino acid sequence of SEQ ID NO: 88. Or (iii) said VL1 comprises the amino acid sequence of SEQ ID NO: 4, said VH1 contained the amino acid sequence of SEQ ID NO: 3, said VL2 contained the amino acid sequence of SEQ ID NO: 2, and said VH2 contained the amino acid sequence of SEQ ID NO: 1. It contains an amino acid sequence;
Or
(II) The first target protein is BAFF and the second target protein is IL-23A.
The VL1 comprises the amino acid sequence of SEQ ID NO: 2, the VH1 comprises the amino acid sequence of SEQ ID NO: 1, the VL2 comprises the amino acid sequence of SEQ ID NO: 4, and the VH2 comprises the amino acid sequence of SEQ ID NO: 3;
The compound. The compound according to claim 8, wherein the first linker and the second linker include the amino acid sequence of SEQ ID NO: 69. The compound according to claim 8 or 9, wherein the third linker and the fourth linker contain the amino acid sequence of SEQ ID NO: 70. The compound according to any one of claims 8 to 10, which comprises one or more of the following characteristics:
(I) The CH2 contains alanine at position 234 and alanine at position 235 according to the EU index similar to Kabat;
(Ii) The amino acid sequences of CH1, the hinge region, CH2, and CH3 are derived from IgG1 or IgG4;
(Iii) The hinge region comprises the amino acid sequence of SEQ ID NO: 76, the amino acid sequence of SEQ ID NO: 130, or the amino acid sequence of SEQ ID NO: 134. The compound according to any one of claims 8 to 10.
(I) The amino acid sequences of the CH1, the hinge region, the CH2, and the CH3 are derived from IgG1, the hinge region contains the amino acid sequence of SEQ ID NO: 76, and the CH2 is the same EU as Kabat. Does the index number contain alanine at position 234 and alanine at position 235; or (ii) the amino acid sequences of CH1, said hinge region, said CH2, and said CH3 are derived from IgG4 and said hinge region. Contains the amino acid sequence of SEQ ID NO: 134;
The compound. The compound according to claim 8.
(A) Does each of the first polypeptides contain the amino acid sequence of SEQ ID NO: 5 and each of the second polypeptides contains the amino acid sequence of SEQ ID NO: 6;
(B) Does each of the first polypeptides contain the amino acid sequence of SEQ ID NO: 15 and each of the second polypeptides contains the amino acid sequence of SEQ ID NO: 16;
(C) Does each of the first polypeptides contain the amino acid sequence of SEQ ID NO: 37 and each of the second polypeptides contains the amino acid sequence of SEQ ID NO: 38;
(D) Does each of the first polypeptides contain the amino acid sequence of SEQ ID NO: 41 and each of the second polypeptides contains the amino acid sequence of SEQ ID NO: 42;
(E) Each of the first polypeptides comprises the amino acid sequence of SEQ ID NO: 45 and each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 46; or (f) the first polypeptide. Each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 49; each of the second polypeptides comprises the amino acid sequence of SEQ ID NO: 50;
The compound. A pharmaceutical composition comprising the compound according to any one of claims 1 to 13. A pharmaceutical composition comprising the compound according to any one of claims 1 to 13, for use in the treatment of autoimmune diseases or inflammatory diseases. A pharmaceutical composition comprising the compound according to any one of claims 1 to 13 for use in treatment. A nucleic acid comprising a nucleotide sequence encoding the polypeptide according to any one of claims 1 to 13. A vector containing the nucleic acid according to claim 17. The vector according to claim 18, further comprising a promoter operably linked to the nucleic acid. A cell comprising the nucleic acid according to claim 17 or a vector containing the nucleic acid. Any one of claims 1 to 13, comprising obtaining a cell containing the nucleic acid encoding the polypeptide according to any one of claims 1 to 13 and expressing the nucleic acid in the cell. The method for producing a polypeptide according to. 21. The method of claim 21, further comprising isolating and purifying the polypeptide. The autoimmune or inflammatory disease is systemic lupus erythematosus (SLE), kidney-related systemic lupus erythematosus / lupus nephritis (LN), ANCA-associated vasculitis (AAV), primary Schegren syndrome (pSS), chronic implants. 15. Claim 15 comprising anti-host disease (cGVHD), systemic lupus erythematosus (SSc), rheumatoid arthritis (RA), psoriatic arthritis (Ps), lupus erythematosus (AS), or psoriatic arthritis (PA) Composition.

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